High-affinity anti-mertk antibodies and uses thereof

ABSTRACT

The present disclosure provides antibodies (e.g., humanized antibodies) that specifically bind to Mer Tyrosine Kinase (MERTK; e.g., human MERTK) and compositions comprising such antibodies. The present disclosure also provides antibody-drug conjugates comprising (i) an anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK), and (ii) cytotoxic agents conjugated directly to the antibodies or conjugated to the antibodies via linkers, and compositions comprising such antibody-drug conjugates. The present disclosure also provides methods for treating cancer, comprising administering to a human subject in need thereof (a) an anti-MERTK antibody that specifically binds to MERTK (e.g., human MERTK) or an antigen-binding fragment thereof described herein, or (b) an antibody-drug conjugate that comprises (i) an anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK), and (ii) a cytotoxic agent conjugated directly to the antibody or conjugated to the antibody via a linker.

This application claims the benefit of U.S. Provisional Application No.62/810,841, filed Feb. 26, 2019, which is incorporated by referenceherein in its entirety.

1. FIELD

This application incorporates by reference a Sequence Listing submittedwith this application as a text file in ASCII format entitled“13256-008-999_SEQ_LISTING.txt” created on Feb. 19, 2020 and having asize of 94,274 bytes.

The present disclosure provides antibodies (e.g., humanized antibodies)that specifically bind to Mer Tyrosine Kinase (MERTK; e.g., human MERTK)and compositions comprising such antibodies. The present disclosure alsoprovides antibody-drug conjugates comprising (i) an anti-MERTK antibodyor antigen-binding fragment thereof described herein that specificallybinds to MERTK (e.g., human MERTK), and (ii) cytotoxic agents conjugateddirectly to the antibodies or conjugated to the antibodies via linkers,and compositions comprising such antibody-drug conjugates. The presentdisclosure also provides methods for treating cancer, comprisingadministering to a human subject in need thereof (a) an anti-MERTKantibody that specifically binds to MERTK (e.g., human MERTK) or anantigen-binding fragment thereof described herein, or (b) anantibody-drug conjugate that comprises (i) an anti-MERTK antibody orantigen-binding fragment thereof that specifically binds to MERTK (e.g.,human MERTK), and (ii) a cytotoxic agent conjugated directly to theantibody or conjugated to the antibody via a linker.

2. BACKGROUND

Mer Tyrosine Kinase (MERTK), also referred to as c-mer, MER,Proto-oncogene c-Mer, Receptor Tyrosine Kinase MerTK, Tyrosine-proteinKinase Mer, STK Kinase, RP38, or MGC 133349, is a member of the TAMfamily of receptor tyrosine kinases, which also include AXL and TYRO3kinases. MERTK transduces signals from the extracellular space viaactivation by binding of ligands, most notably Gas-6, a soluble protein.Gas-6 binding to MERTK induces autophosphorylation of MERTK on itsintracellular domain, resulting in downstream signal activation(Cummings C T et al., (2013) Clin Cancer Res 19: 5275-5280; Verma A etal., (2011) Mol Cancer Ther 10: 1763-1773).

MERTK exists in both membrane bound and soluble forms. The extracellulardomain can be cleaved to generate a soluble extracellular domain, whichis hypothesized to act as a decoy receptor to negatively regulate MERTKreceptor activation on cells by reducing the ability and/or availabilityof soluble Gas-6 ligand to bind membrane-bound MERTK (Sather S et al.,(2007) Blood 109: 1026-1033). As a result MERTK has dual roles relatedto cancer progression, angiogenesis, and metastasis. On the one hand,Gas-6 activation of MERTK on endothelial cells results in inhibition ofendothelial cell recruitment by cancer cells in a co-culture system.Endothelial recruitment is a key feature of cancer cells that allows fortumor angiogenesis, tumor growth, and metastasis. Downregulation ofMERTK on metastatic breast cancer cells was also shown to inhibitendothelial recruitment (Png K J et al., (2012) Nature 481: 190-194).However, on the other hand, MERTK plays an opposite role in cancercells, where its over-expression leads to increased metastasis, likelyby releasing cleaved MERTK to generate soluble MERTK extracellulardomain protein as a decoy receptor. Additionally, ligand-dependent MERTKactivation on cancer cells stimulates oncogenic pathways, including theAKT pathway (Linger R M, Cohen R A, Cummings C T, Sather S,Migdall-Wilson J, Middleton D H, Lu X, Barón A E, Franklin W A, MerrickD T, Jedlicka P, DeRyckere D, Heasley L E, Graham D K. Mer or Axlreceptor tyrosine kinase inhibition promotes apoptosis, blocks growthand enhances chemosensitivity of human non-small cell lung cancer(Oncogene. 2013 Jul. 18; 32(29):3420-31). Thus, tumor cells overexpressMERTK to promote oncogenic signaling. Also, tumors cells secrete asoluble form of the extracellular MERTK receptor that acts as a decoyreceptor to reduce the ability (and/or availability) of soluble Gas-6ligand to activate MERTK on endothelial cells, ultimately leading toendothelial recruitment, angiogenesis, and cancer progression (Png K Jet al., (2012) Nature 481: 190-194).

MERTK is also involved in modulating macrophage homeostasis and themaintenance of anti-inflammatory conditions. On macrophages, MERTKmediates phagocytotic clearance of apoptotic cells and the release ofIL-10 and acute inflammatory cytokines, whose expression are stimulatedby Gas6-dependent binding to MERTK (Zizzo et al., (2012) J. ImmunologyOctober 1; 189(7):3508-20; Cook et al., J Clin Invest. 2013;123(8):3231-3242).

Historically, there have been efforts to generate inhibitors, of MERTKfor the treatment of cancer (e.g., compound UNC 1062, a potent smallmolecule MERTK inhibitor developed as an anticancer compound), becauseMERTK was thought to solely function as an oncogene (Liu J et al.,(2013) Eur J Med Chem 65: 83-93; Cummings C T et al., (2013) Clin CancerRes 19: 5275-5280; Verma A et al., (2011) Mol Cancer Ther 10:1763-1773). In recent years, antibody-drug conjugates (ADCs) have becomeone of the fastest growing classes of cancer therapeutics (Beck A etal., (2017) Nat Rev Drug Discov 16: 315-337; Peters C and Brown S,(2015) Biosci Rep 35: art:e00225).

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present disclosure.

3. SUMMARY

Disclosed herein are antibodies and antigen-binding fragments thereofthat specifically bind to MERTK (e.g., human MERTK) with high affinity.In a specific embodiment of any of the antibodies or antigen-bindingfragments thereof described herein, the antibody specifically recognizesthe extracellular domain of MERTK (e.g., human MERTK), and theextracellular domain comprises the amino acid sequence of SEQ ID NO: 132

In a specific embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof specifically binds to MERTK (e.g., human MERTK) andleads to internalization of MERTK from the cell surface. In a specificembodiment, an anti-MERTK antibody or antigen-binding fragment thereofblocks Gas-6 induced AKT phosphorylation. In another specificembodiment, an anti-MERTK antibody or an antigen-binding fragmentthereof prevents MERTK activation. In another specific embodiment, ananti-MERTK antibody or antigen-binding fragment thereof described hereinspecifically recognizes the extracellular portion of human MERTK. Inanother specific embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof reduces colony formation by cancer cells. In aparticular embodiment, an anti-MERTK antibody or antigen-bindingfragment described herein is monoclonal. In another particularembodiment, an anti-MERTK antibody or antigen-binding fragment thereofdescribed herein is an immunoglobulin comprising two identical lightchains and two identical heavy chains. In a specific embodiment, ananti-MERTK antibody is a humanized antibody.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), comprises a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 105.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises a heavy chain variableregion (VH) comprising the amino acid sequence of SEQ ID NO: 107.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises a heavy chain variableregion (VH) comprising the amino acid sequence of SEQ ID NO: 108.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK) comprises a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 109.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises a heavy chain variableregion (VH) comprising the amino acid sequence of SEQ ID NO: 110.

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), comprises a light chain variable region (VL)comprising the amino acid sequence of SEQ ID NO: 106. In anotherparticular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), comprises a light chain variable region (VL)comprising the amino acid sequence of SEQ ID NO: 111.

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 105and (b) a VL comprising the amino acid sequence of SEQ ID NO: 106. Inanother embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 107and (b) a VL comprising the amino acid sequence of SEQ ID NO: 106. Inanother embodiment, an anti-MERTK antibody or the antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 108and (b) a VL comprising the amino acid sequence of SEQ ID NO: 106. Inanother embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof which specifically binds to MERTK (e.g. human MERTK),comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 109and (b) a VL comprising the amino acid sequence of SEQ ID NO: 106. Inanother embodiment, an anti-MERTK antibody or antigen-binding fragmentthereof, which specifically binds to MERTK (e.g., human MERTK),comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 110and (b) a VL comprising the amino acid sequence of SEQ ID NO: 111.

In certain embodiments, an antibody provided herein, which specificallybinds to MERTK (e.g., human MERTK), comprises heavy chain constantregions, or light chain constant regions, or both. In some embodiments,the heavy chain constant region is selected from the group of humanimmunoglobulins consisting of IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Incertain embodiments, the light chain constant region is selected fromthe group of human immunoglobulins consisting of IgGκ and IgGλ. In someembodiments, the antibody comprises a constant region having increasedbinding affinity to one or more human Fc gamma receptor(s).

In some embodiments, the antibody comprises a constant region havingdecreased binding affinity to one or more human Fc gamma receptor(s).

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH),which comprises the amino acid sequence of SEQ ID: 105. In one furtherembodiment of such a specific embodiment, each of the light chainscomprises a variable region (VL), which comprises the amino acid of SEQID NO: 106

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)which comprises the amino acid of SEQ ID NO: 107. In one furtherembodiment of such a specific embodiment, each of the light chainscomprises a variable region (VL) comprising the amino acid sequence ofSEQ ID NO: 106.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)comprising the amino acid sequence of SEQ ID NO: 108. In one furtherembodiment of such a specific embodiment, each of the light chainscomprises a variable region (VL), comprising the amino acid sequence ofSEQ ID NO: 106

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)comprising the amino acid sequence of SEQ ID NO: 110. In one furtherembodiment of such a specific embodiment, each of the light chainscomprises a variable region (VL) comprising the amino acid sequence ofSEQ ID: 111.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)comprising the amino acid sequence of SEQ ID NO: 109. In one furtherembodiment of such a specific embodiment, each of the light chainscomprises a variable region (VL) comprising the amino acid sequence ofSEQ ID: 106.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the light chain comprises a variable region (VL), whichcomprises SEQ ID NO: 106.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the light chain comprises a variable region (VL) whichcomprises SEQ ID NO: 111.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the heavy chain comprises a variable region (VH), whichcomprises SEQ ID NO: 105.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the heavy chain comprises a variable region (VH), whichcomprises SEQ ID NO: 107.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the heavy chain comprises a variable region (VH), whichcomprises SEQ ID NO: 108.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the heavy chain comprises a variable region (VH) whichcomprises SEQ ID NO: 109.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the heavy chain comprises a variable region (VH), whichcomprises SEQ ID NO: 110.

In a specific embodiment of any of the antibodies or antigen-bindingfragments thereof provided herein, wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the antibody or antigen-binding fragment thereof comprisesa human-derived constant region. In one further embodiment of such aspecific embodiment, the heavy chain constant region has an isotypeselected from the group consisting of gamma1, gamma2, gamma3, andgamma4.

In a specific embodiment of any of the antibodies or antigen-bindingfragments thereof provided herein, the antibody binds MERTK (e.g., humanMERTK) on cells with an EC50 in the range of about 1 nM to 15 nM (e.g.,6.5 nM to 8 nM.)

In a specific embodiment of any of the antibodies or antigen-bindingfragments thereof provided herein, the antibody or antigen-bindingfragment decreases the expression level of MERTK on cancer cells (e.g.the melanoma cell line SKMEL5). In another specific embodiment of any ofthe antibodies or antigen-binding fragments thereof provided herein, theantibody or antigen-binding fragment decreases the expression level ofMERTK on human M2 macrophages. In another specific embodiment of any ofthe antibodies or antigen-binding fragments thereof provided herein, theantibody or antigen-binding fragment decreases the level of MERTK on invitro differentiated human M2 macrophages.

In a specific embodiment of any of the antibodies or antigen-bindingfragments thereof provided herein, the antibody or antigen-bindingfragment thereof comprises a human-derived constant region. In onefurther embodiment of such a specific embodiment, the antibody orantigen-binding fragment thereof is a humanized immunoglobulin.

In a specific embodiment of any of the antibodies or antigen-bindingfragments thereof provided herein, wherein the antibody is not amonoclonal antibody or an immunoglobulin comprising two identical lightchains and two identical heavy chains, the antibody or antigen-bindingfragment thereof is a bispecific antibody.

In another aspect, provided herein is an immunoglobulin thatspecifically binds to MERTK (e.g., human MERTK), comprising (i) a heavychain variable region that comprises the amino acid sequence of SEQ IDNO: 105, and (ii) a light chain variable region that comprises the aminoacid sequence of SEQ ID NO: 106.

In another aspect, provided herein is an immunoglobulin thatspecifically binds to MERTK (e.g., human MERTK), comprising (i) a heavychain variable region that comprises the amino acid sequence of SEQ IDNO: 107, and (ii) a light chain variable region that comprises the aminoacid sequence of SEQ ID NO: 106.

In another aspect provided herein is an immunoglobulin that specificallybinds to MERTK (e.g., human MERTK) comprising (i) a heavy chain variableregion that comprises the amino acid sequence of SEQ ID NO: 108, and(ii) a light chain variable region that comprises the amino acid of SEQID NO: 106.

In another aspect, provided herein is an immunoglobulin thatspecifically binds to MERTK (e.g., human MERTK) comprising (i) a heavychain variable region that comprises the amino acid sequence of SEQ IDNO: 109, and (ii) a light chain variable region that comprises the aminoacid sequence of SEQ ID NO: 106.

In another aspect, provided herein is a humanized immunoglobulin thatspecifically binds to MERTK (e.g., human MERTK), comprising:

-   -   (A) (i) a heavy chain variable region that comprises the amino        acid sequence of SEQ ID NO: 105, and (ii) a light chain variable        region that comprises the amino acid sequence of SEQ ID NO: 106;        or    -   (B) (i) a heavy chain variable region that comprises the amino        acid sequence of SEQ ID NO: 107, and (ii) a light chain variable        region that comprises the amino acid sequence of SEQ ID NO: 106,        or    -   (C) (i) a heavy chain variable region that comprises the amino        acid sequence of SEQ ID NO: 108, and (ii) a light chain variable        region that comprises the amino acid sequence of SEQ ID NO: 106,        or    -   (D) (i) a heavy chain variable region that comprises the amino        acid sequence of SEQ ID NO: 109, and (ii) a light chain variable        region that comprises the amino acid sequence of SEQ ID NO: 106.

In some embodiments, the antibody provided herein is an IgG. In someembodiments, antigen-binding fragment provided herein is an Fab, F(ab′)2or scFv fragment.

In another aspect, provided herein is a chimeric antibody orantigen-binding fragment thereof which binds to MERTK (e.g., humanMERTK). In some embodiments, the chimeric antibody comprises a heavychain variable region (VH) comprising an amino acid of SEQ ID NO: 110.In some embodiments, the chimeric antibody comprises a variable lightchain region (VL) comprising an amino acid of SEQ ID NO: 111. In aspecific embodiment, the chimeric antibody comprises a VH of SEQ ID NO:110 and a VL of SEQ ID NO: 111.

In another specific embodiment, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 112, 114, 115, 116 or117. In another specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof, which specifically binds MERTK (e.g.human MERTK) comprises a light chain comprising the amino acid of SEQ IDNO: 113 or 118. In another specific embodiment, an anti-MERTK antibodycomprises:

-   -   (A) a heavy chain comprising the amino acid of SEQ ID NO: 112        and a light chain comprising the amino acid sequence of SEQ ID        NO: 113; or    -   (B) a heavy chain comprising the amino acid of SEQ ID NO: 114        and a light chain comprising the amino acid sequence of SEQ ID        NO: 113; or    -   (C) a heavy chain comprising the amino acid of SEQ ID NO: 115        and a light chain comprising the amino acid sequence of SEQ ID        NO: 113; or    -   (D) a heavy chain comprising the amino acid of SEQ ID NO: 116        and a light chain comprising the amino acid sequence of SEQ ID        NO: 113; or    -   (E) a heavy chain comprising the amino acid of SEQ ID NO: 117        and a light chain comprising the amino acid sequence of SEQ ID        NO: 113.

In certain embodiments, an anti-MERTK antibody is a bispecific antibody,which comprises two different antigen binding regions, wherein onebinding region specifically binds to MERTK (e.g., human MERTK) and theother binding region binds to an antigen of interest (e.g., an immunecell receptor, a checkpoint receptor, or a tumor-associated antigen).

In specific embodiments, the binding region that binds to MERTKcomprises the variable regions of the z10, z11 or z13 antibody describedherein (e.g. those set forth in Tables 11, 12 and 13 infra). In somespecific embodiments, the other binding region of the bispecificantibody binds to CD3. In other specific embodiments, the other bindingregion of the bispecific antibody binds to PD-L1. In other specificembodiments, the other binding region of the bispecific antibody bindsto LRP1. In other specific embodiments, the other binding region of thebispecific antibody binds to LPR8. In other specific embodiments, theother binding region of the bispecific antibody binds to TGF-β. In otherspecific embodiments, the other binding region of the bispecificantibody binds to ICOS. In other specific embodiments, the other bindingregion of the bispecific antibody binds to CD40. In other specificembodiments, the other binding region of the bispecific antibody bindsto NKGD2. In other specific embodiments, the other binding region of thebispecific antibody binds to TIGIT. In a particular embodiment, thebinding region that binds to CD40 is an agonistic antibody. In anotherparticular embodiment, the binding region that binds to ICOS is anagonistic antibody. In another particular embodiment, the binding regionthat binds to PD-L1 is a blocking antibody.

In another embodiment, provided herein is an anti-MERTK antibody orantigen-binding fragment thereof (e.g, a humanized or human antibody orantigen-binding fragment thereof) that binds to the same epitope ofMERTK (e.g., human MERTK) as an anti-MERTK antibody described herein. Inanother embodiment, provided herein is an anti-MERTK antibody orantigen-binding fragment thereof (e.g, a humanized or human antibody orantigen-binding fragment thereof) that competes with an anti-MERTKantibody or an antigen-binding fragment thereof described herein, forbinding to MERTK (e.g., human MERTK). In another specific embodiment,provided herein is a first anti-MERTK antibody or antigen-bindingfragment thereof that competes with an anti-MERTK antibody or anantigen-binding fragment thereof described herein for binding to MERTK(e.g., human MERTK), wherein the competition is exhibited as reducedbinding of the first anti-MERTK antibody or antigen-binding fragmentthereof to MERTK (e.g., human MERTK) by more than 80% (e.g., 85%, 90%,95%, or 98%, or between 80% to 85%, 80% to 90%, 85% to 90%, or 85% to95%) in the presence of the anti-MERTK antibody or antigen-bindingfragment thereof described herein. In a specific embodiment, theanti-MERTK antibody or antigen-binding fragment thereof that binds tothe same epitope as MERTK (or competes with binding to an anti-MERTKantibody or antigen-binding fragment thereof) is not a murine antibodyor antigen-binding fragment thereof.

In certain embodiments, an anti-MERTK antibody provided herein, whichspecifically binds to MERTK (e.g., human MERTK) with an EC₅₀ in therange of about 1 nM to 10 nM in an assay such as described herein. Insome embodiments, an anti-MERTK antibody or antigen-binding fragmentthereof, which specifically binds to human MERTK. In a specificembodiment, the antibody or antigen-binding fragment thereof providedherein does not bind or does not appreciably bind to human Axl, humanTyro3 or murine MERTK as assessed by an assay described therein or knownto one of skill in the art. In some embodiments, an anti-MERTK antibodyor an antigen-binding fragment thereof binds to human MERTK andcynomolgus monkey MERTK. In specific embodiments, an antibody providedherein is isolated. In specific embodiments, an antibody provided hereinis a monoclonal antibody. In a specific embodiment, an anti-MERTKantibody or antigen-binding fragment thereof provided herein is bivalentand is capable of binding to two MERTK molecules (e.g. human MERTKmolecules).

In another aspect, provided herein are polynucleotide sequences encodingan antibody or an antigen-binding fragment thereof. In a specificembodiment, provided herein is a polynucleotide sequence comprising anucleic acid molecule that encodes a heavy chain variable region,wherein the nucleic acid molecule comprises the nucleic acid sequence ofSEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123. Inanother specific embodiment, provided herein is a polynucleotidesequence comprising a nucleic acid molecule that encodes a light chainvariable region, wherein the nucleic acid molecule comprises the nucleicacid sequence of SEQ ID NO: 120 or SEQ ID NO: 124. In specificembodiments, the nucleic acid molecule or polynucleotide sequence isisolated.

In another aspect, provided herein are vectors comprising apolynucleotide sequence encoding an antibody or an antigen-bindingfragment described herein. In certain embodiments, a vector (e.g., anisolated vector) comprises a polynucleotide encoding a heavy chainvariable region and/or a light chain variable region, or a heavy chainand/or a light chain of an anti-MERTK antibody described herein. Incertain embodiments, a host cell comprises the polynucleotide or vector.Examples of host cells include E. coli, Pseudomonas, Bacillus,Streptomyces, yeast, 293F, CHO, YB/20, NS0, PER-C6, HEK-293, HEK-293T,NIH-3T3, HeLa, BHK, Hep G2, SP2/0, R1.1, B-W, L-M, COS 1, COS 7, BSC1,BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissueculture. In a specific embodiment, provided herein is a method ofproducing an anti-MERTK antibody or antigen-binding fragment thereofthat specifically binds to MERTK (e.g., human MERTK) comprisingculturing a host cell so that the polynucleotide is expressed and theantibody is produced.

In another aspect, provided herein is an ex vivo cell containing one ormore polynucleotides encoding of any of the antibodies orantigen-binding fragments thereof described herein, or the any of theimmunoglobulins described herein. In a specific embodiment, an ex vivocell contains one or more polynucleotides encoding an antibody selectedfrom the group consisting of:

-   -   (a) a first immunoglobulin comprising (i) a heavy chain variable        region that comprises the amino acid sequence of SEQ ID NO: 105,        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 106;    -   (b) a second immunoglobulin comprising (i) a heavy chain        variable region that comprises the amino acid sequence of SEQ ID        NO: 107, and (ii) a light chain variable region that comprises        the amino acid sequence of SEQ ID NO: 106; and    -   (c) a third immunoglobulin comprising (i) a heavy chain variable        region that comprises the amino acid sequence of SEQ ID NO: 108,        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 106;    -   (d) a fourth immunoglobulin comprising (i) a heavy chain region        that comprises the amino acid sequence of SEQ ID NO: 109        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 106; and    -   (e) a fifth immunoglobulin comprising (i) a heavy chain variable        region that comprises the amino acid sequence of SEQ ID NO: 110,        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 111.

In another aspect, provided herein is an ex vivo cell containing apolynucleotide encoding any of the antibody heavy chains describedherein.

In another aspect, provided herein is an ex vivo cell containing apolynucleotide encoding any of the antibody light chains describedherein. In another aspect, provided herein is a method of producing anantibody or antigen-binding fragment thereof, comprising culturing an exvivo cell containing one or more polynucleotides encoding any of theantibodies or antigen-binding fragments described herein, underconditions such that the one or more polynucleotides are expressed bythe cell to produce the antibody or the antigen-binding fragment thereofencoded by the polynucleotides. In a specific embodiment a method ofproducing an antibody or antigen-binding fragment thereof comprisesculturing an ex vivo cell containing one or more polynucleotidesencoding an antibody selected from the group consisting of:

-   -   (a) a first immunoglobulin comprising (i) a heavy chain variable        region that comprises the amino acid sequence of SEQ ID NO: 105,        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 106;    -   (b) a second immunoglobulin comprising (i) a heavy chain        variable region that comprises the amino acid sequence of SEQ ID        NO: 107, and (ii) a light chain variable region that comprises        the amino acid sequence of SEQ ID NO: 106;    -   (c) a third immunoglobulin comprising (i) a heavy chain variable        region that comprises the amino acid sequence of SEQ ID NO: 108,        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 106;    -   (d) a fourth immunoglobulin comprising (i) a heavy chain region        that comprises the amino acid sequence of SEQ ID NO: 109        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 106; and    -   (e) a fifth immunoglobulin comprising (i) a heavy chain variable        region that comprises the amino acid sequence of SEQ ID NO: 110,        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 111,        under conditions such that the one or more polynucleotides are        expressed by the cell to produce the antibody encoded by the        polynucleotide.

In another aspect, provided herein is a method of producing an antibodyheavy chain, comprising culturing an ex vivo cell containing apolynucleotide encoding any of the antibody heavy chains describedherein under conditions such that the polynucleotide is expressed by thecell to produce the antibody heavy chain encoded by the polynucleotide.

In another aspect, provided herein is a method of producing an antibodylight chain, comprising culturing an ex vivo cell containing apolynucleotide encoding any of the antibody light chains describedherein under conditions such that the polynucleotide is expressed by thecell to produce the antibody light chain encoded by the polynucleotide.

In another aspect, provided herein are antibody-drug conjugatescomprising: (a) an antibody moiety described herein that is an antibodyor antigen-binding fragment thereof that specifically binds MERTK (e.g.,human MERTK); (b) one or more drug moieties, each drug moiety being acytotoxic agent; and (c) optionally a linker; wherein the cytotoxicagent is conjugated directly to the antibody moiety or is conjugated tothe antibody moiety via the linker.

In a specific embodiment, the molar ratio of the antibody moiety to thedrug moiety is between 1:1 and 1:12.

In a specific embodiment, the molar ratio of the antibody moiety to thedrug moiety is between 1:1 and 1:8.

In another specific embodiment, the molar ratio of the antibody moietyto the drug moiety is between 1:3 and 1:5.

In another specific embodiment, the molar ratio of the antibody moietyto the drug moiety is between 1:9.

In a specific embodiment, the antibody-drug conjugate comprises thelinker and the linker is a cleavable linker.

In another specific embodiment, the antibody-drug conjugate comprisesthe linker and the linker is a non-cleavable linker.

In another specific embodiment, the antibody-drug conjugate comprisesthe linker and the linker ismaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (also knownas “mc-vc-PABC”) (for the structure of mc-vc-PABC; see FIG. 13A). Inanother specific embodiment, the antibody-drug conjugate comprises thelinker and the linker is CL2 (for the structure of CL2, see FIG. 13B andCardillo et al. 2011, Clin Cancer Res; 17(10); 3157-69. In anotherspecific embodiment, the antibody-drug conjugate comprises the linkerand the linker is CL2A (for the structure of CL2A, see FIG. 13B andGoldberg et al. 2018, Oncotarget; 9(48); 28989-29006, and Cardillo etal. 2011, Clin Cancer Res; 17(10); 3157-69).

In specific embodiments, the cytotoxic agent is a small molecule, anucleotide, a peptide, or a non-antibody protein. In one furtherembodiment of such a specific embodiment, the cytotoxic agent is a smallmolecule.

In another specific embodiment, the cytotoxic agent is an auristatin, amaytansinoid, a pyrrolobenzodiazepine, an indolinobenzodiazepine, acalicheamicin, a camptothecin analogue, a duocarmycin, a tubulininhibitor, a tubulysin or tubulysin analogue, amberstatin269,doxorubicin, an antibiotic, an anthracycline, a microtubule inhibitor, aspliceostatin, or a thailanstatin. In one further embodiment of such aspecific embodiment, the cytotoxic agent is monomethyl auristatin E(MMAE) or monomethyl auristatin F (MMAF). In another further embodimentof such a specific embodiment, the cytotoxic agent is DM1 or DM4. Inanother further embodiment of such a specific embodiment, the cytotoxicagent is SN-38.

In another aspect, provided herein is a method of producing anantibody-drug conjugate described herein wherein the linker is notpresent, the method comprising: (a) conjugating the cytotoxic agentdirectly to the antibody moiety to produce the antibody-drug conjugate;and (b) purifying the antibody-drug conjugate.

In another aspect, provided herein is a method of producing anantibody-drug conjugate described herein wherein the antibody-drugconjugate comprises the linker, the method comprising the followingsteps in the order stated: (a) conjugating the linker directly to theantibody moiety to produce a linker-antibody moiety; (b) conjugating thelinker of the linker-antibody moiety directly to the cytotoxic agent toproduce the antibody-drug conjugate; and (c) purifying the antibody-drugconjugate.

In another aspect, provided herein is a method of producing anantibody-drug conjugate described herein, wherein the antibody-drugconjugate comprises the linker, the method comprising the followingsteps in the order stated: (a) conjugating the linker directly to thecytotoxic agent to produce a linker-cytotoxic agent moiety; (b)conjugating the linker of the linker-cytotoxic agent moiety directly tothe antibody moiety to produce the antibody-drug conjugate; and (c)purifying the antibody-drug conjugate.

In another aspect, provided herein are pharmaceutical compositionscomprising an anti-MERTK antibody or antigen-binding fragment thereofdescribed herein, or an antibody-drug-conjugate described herein. In aspecific embodiment, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of any of the antibodiesor antigen-binding fragments described herein or any of theantibody-drug conjugates described herein. In a specific embodiment, apharmaceutical composition comprises:

-   -   (a) a first immunoglobulin comprising (i) a heavy chain variable        region that comprises the amino acid sequence of SEQ ID NO: 105,        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 106;    -   (b) a second immunoglobulin comprising (i) a heavy chain        variable region that comprises the amino acid sequence of SEQ ID        NO: 107, and (ii) a light chain variable region that comprises        the amino acid sequence of SEQ ID NO: 106;    -   (c) a third immunoglobulin comprising (i) a heavy chain variable        region that comprises the amino acid sequence of SEQ ID NO: 108,        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 106,    -   (d) a fourth immunoglobulin comprising (i) a heavy chain region        that comprises the amino acid sequence of SEQ ID NO: 109        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 106; or    -   (e) a fourth immunoglobulin comprising (i) a heavy chain        variable region that comprises the amino acid sequence of SEQ ID        NO: 110, and (ii) a light chain variable region that comprises        the amino acid sequence of SEQ ID NO: 106,        and a pharmaceutically acceptable carrier.

In some embodiments, a pharmaceutical composition comprises apolynucleotide sequence comprising a nucleic acid sequence encoding anantibody or an antigen-binding fragment thereof.

In another aspect, provided herein are methods of treating cancer in asubject in need thereof, comprising administering to said subject ananti-MERTK antibody or an antigen-binding fragment thereof describedherein, or an antibody-drug conjugate described herein. In a specificembodiment, provided herein is a method of treating cancer in a subjectin need thereof, comprising administering to said subject any of thepharmaceutical compositions described herein.

In some embodiments, a tumor sample from the subject is assessed foroverexpression of phosphorylated MERTK prior to treatment in accordancewith the methods described herein. In certain embodiments, the subjectis treated if the tumor sample overexpresses phosphorylated MERTK.

In a specific embodiment of the preceding aspect of a method of treatingcancer, the cancer is a cancer of the head and neck, lung, breast, bone,ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid,biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,bladder, prostate or thyroid. In a certain embodiment of such a specificembodiment, the cancer is breast cancer. In a further embodiment of sucha certain embodiment, the cancer is triple-negative breast cancer.

In a specific embodiment of the preceding aspect of a method of treatingcancer, the cancer is a sarcoma, squamous cell carcinoma, melanoma,glioma, glioblastoma, neuroblastoma, gastric cancer, colorectal cancer,non-small cell lung carcinoma or Kaposi's sarcomas. In a specificembodiment of the aspect of a method of treating cancer, the cancer is aleukemia or lymphoma. In a further specific embodiment, the cancer isacute myelogenous leukemia. In another further specific embodiment, thecancer is acute lymphocytic leukemia. In another further embodiment, thecancer is multiple myeloma.

In a specific embodiment, a cancer treated in accordance with themethods described herein overexpresses MERTK, is associated withconstitutively active MERTK (e.g., human MERTK), or both. In anotherspecific embodiment, cancerous cells of the cancer overexpressphosphorylated MERTK.

In a specific embodiment of the method of treating cancer, the methodfurther comprises administering to the subject an additional therapeuticagent.

In a specific embodiment of the embodiment wherein the method furthercomprises administering to the subject an additional therapeutic agent,the additional therapeutic agent is for treating the cancer. Theadditional therapeutic agent may be administered in the samepharmaceutical composition or in a different pharmaceutical compositionthan the anti-MERTK antibody or an antigen-binding fragment thereof, oran antibody-drug conjugate.

In a specific embodiment of the embodiment wherein the additionaltherapeutic agent is for treating the cancer, the additional therapeuticagent is an agent used to treat breast cancer, an agent used to treatmelanoma, an immunotherapy, or an angiogenesis inhibitor. In one furtherembodiment of such a specific embodiment, the additional therapeuticagent is an agent used to treat breast cancer that is selected from thegroup consisting of Tamoxifen, Raloxifene, Paclitaxel, Cyclophosphamide,Docetaxel, Vinblastine, Fluorouracil, Everolimus, Trastuzumab,Trastuzumab-Emtansine, Pertuzumab, and Lapatinib Ditosylate. In anotherfurther embodiment of such a specific embodiment, the additionaltherapeutic agent is an agent used to treat melanoma that is selectedfrom the group consisting of a BRAF inhibitor, a MEK inhibitor, andDacarbazine. In another further embodiment of such a specificembodiment, the additional therapeutic agent is an agent that blocksimmune checkpoint signaling. In another further embodiment, theadditional therapeutic agent is an anti-CTLA-4 antibody, an anti-PD-1antibody, or an anti-PD-L1 antibody. In another further embodiment ofsuch a specific embodiment, the additional therapeutic agent is anangiogenesis inhibitor that is selected from the group consisting of aVEGF inhibitor, a VEGFR2 inhibitor, Sunitinib, and Sorafenib.

In a specific embodiment of the method of treating cancer, the subjectis a human. In a specific embodiment, provided herein is anantibody-drug conjugate comprising an antibody moiety comprising a heavychain variable region (VH) and a light chain variable region (VL),wherein the VH comprises the amino acid sequence of sequence of SEQ IDNO: 105 and the VL comprises the amino acid sequence of SEQ ID NO: 106,wherein the antibody moiety is linked to MMAE via the mc-vc-PABC linker.In another specific embodiment, provided herein is an antibody-drugconjugate comprising an antibody moiety comprising a heavy chainvariable region (VH) and a light chain variable region (VL), wherein theVH comprises the amino acid sequence of sequence of SEQ ID NO: 105 andthe VL comprises the amino acid sequence of SEQ ID NO: 106, wherein theantibody moiety is linked to SN-38 via the CL2A linker.

In certain embodiments, the antibody-drug conjugate comprises anantibody moiety that is an immunoglobulin. In some embodiments, theantibody-drug conjugate comprises an antibody moiety wherein theantibody moiety is and immunoglobulin and the immunoglobulin comprises ahuman constant region.

4. BRIEF DESCRIPTIONS OF FIGURES

FIG. 1. Determination of off-rates (K_(d)) for humanized anti-MERTKantibodies and a chimeric anti-MERTK antibody using surface plasmonresonance (SPR) measurements.

FIG. 2. Protein purification summary for three humanized anti-MERTKantibodies (z10, z11, and z13) and a chimeric anti-MERTK antibody (xAb).

FIG. 3A-FIG. 3B. FIG. 3A. Flow cytometry analysis of the z10 antibodybinding affinity for human MERTK expressed by melanoma cells. FIG. 3B.SPR measurements of the binding of the z10 antibody to humanextracellular MERTK.

FIGS. 4A-4D. The z10 antibody specifically binds to human MERTK (FIG.4A), as measured in an affinity binding assay using ELISA. However, thez10 antibody does not bind to the extracellular domain of human Ax1(FIG. 4B), human Tyro 3 (FIG. 4C) or murine MERTK (FIG. 4D), as assessedin an affinity binding assay using ELISA. The extracellular domains ofhuman MERTK, human Axl and murine MERTK were conjugated to an IgG Fcdomain and used in the ELISA.

FIGS. 5A-5B. The z10 antibody induces human MERTK degradation on cancercells (specifically SKMEL5 cells). SKMEL5 cells were incubated with z10antibody at various concentrations for 24 hours (FIG. 5A) or with 0.3μg/mL of the antibody for various time periods (FIG. 5B) before thelevel of human MERTK was assessed by Western Blot analysis.

FIGS. 6A-6B. The z10 antibody degrades human MERTK on in vitrodifferentiated human M2 macrophages. In vitro M2 macrophages weretreated with 0.3 μg/mL of the z10 antibody or control IgG, and the cellswere incubated for various periods of time before the level of humanMERTK was assessed by Western Blot analysis. FIG. 6A provides theWestern Blot results and FIG. 6B provides the relative quantification ofhuman MERTK to tubulin.

FIGS. 7A-7B. Human MERTK degradation is mediated via internalization ofhuman MERTK of SKMEL5 cells. SKMEL5 cells were incubated with controlIgG or the z10 antibody, both labeled with pHrodo, which is onlymeasureable in low pH conditions e.g. upon uptake into lysosomes.Surface human MERTK was measured using a commercially available MERTKantibody. FIG. 7A shows the fluorescence over time and FIG. 7B arerepresentative images.

FIGS. 8A-8C. The z10 antibody blocks Gas6-induced phosphorylation of AKTin human cancer cells. SKMEL5 cells were incubated with 0.3 l/mL of thez10 antibody or control IgG for 2 hours before being incubated with 200nM of Gas6 for 10 minutes. FIG. 8A shows a Western Blot result, FIG. 8Bshows the quantity of human MERTK relative to tubulin, and FIG. 8C showsthe quantity of phosphorylated AKT to tubulin.

FIGS. 9A-9D. The z10 antibody inhibits colony formation of human cancercells. The figure shows the reduction in the number of colonies observedafter 500 SKMEL5 cells were cultured for 12 days in the presence of 0.3μg/mL or 1 μg/mL of the z10 antibody relative to the number of coloniesobserved when 500 SKMEL5 cells were cultured for 12 days with controlIgG. FIG. 9A shows colony formation of cancer cells after 12 days ofincubation with 0.3 g/mL of the z10 antibody or control IgG antibody.FIG. 9B shows colony formation of cancer cells after 12 days ofincubation with 1 g/mL of the z10 antibody or control IgG antibody. FIG.9C shows numbers of colonies formed after 12 days of incubation with 0.3μg/mL of the z10 antibody or control IgG antibody. FIG. 9D shows numbersof colonies formed after 12 days of incubation with 1 μg/mL of the z10antibody or control IgG antibody.

FIG. 10. The z10 antibody induces cytokine responses in M2 macrophagesand CD14+ monocytes.

FIGS. 11A and 11B. z10 inhibits cancer cell survival in vitro. 1,000RPMI8226 multiple myeloma cells (FIG. 11A) or 200 SKMEL5 melanoma cells(FIG. 11B) were cultured in the presence of either IgG control or z10.On day 6 (RPMI8226) or day 4 (SKMel5), viability was assessed usingCellTiter-Glo 2.0 Cell Viability Assay.

FIGS. 12A and 12B. MDA-MB-231-LM2: z10 Naked Antibody Anti-tumorefficacy. 50,000 MDA-MB-231-LM2 TNBC cells were injected into the tailvein of NSG mice. Treatment started on the day of tumor cellinoculation, twice per week i.p. for the duration of the study. FIG. 12Ashows lung colonization of MDA-MB-231 LM2 triple negative breast cancercells using IVIS imaging. FIG. 12B quantifies the lung colonization ofMDA-MB-231 LM2 triple negative breast cancer cells. * designates ap-value of less than 0.05.

FIGS. 13A and 13B. FIG. 13A shows the structure ofmaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethylauristatin E (mc-vc-PABC-MMAE) conjugated to an antibody (Ab). FIG. 13Bshows the structure of SN-38 conjugated to an antibody (Ab) via a CL2 orCL2A linker. “Ab” represents an antibody, which by way of example butnot limitation can be z10 or a control IgG. The CL2 linker has aCathepsin B cleavage site (see FIG. 13B when AA isphenylalanine-lysine), while the CL2A linker has no Cathepsin B cleavagesite (see FIG. 13B when AA is lysine).

FIGS. 14A and 14B show the results of size-exclusion high pressureliquid chromatography, measuring the purity of z10 conjugated to MMAEvia a mc-vc-PABC linker (z10-MMAE, see FIG. 13A where Ab is z10) (FIG.14A) and z10 conjugated to SN-38 via a CL2A linker (z10-SN-38, see FIG.13B where Ab is z10 and AA is lysine) (FIG. 14B).

FIG. 15: z10-ADCs bind to MERTK with a Kd similar to z10. FIG. 15provides z10-ADC binding affinity data. hMER recombinant protein (Mer-Fcconjugate) (50 nM-0.05 nM) was equilibrated with 0.3 nM z10, z10-MMAE orz10-SN-38, respectively before addition to ELISA plates pre-coated withhMER recombinant protein. Antibody binding was detected usingAP-conjugated anti-human IgG secondary antibody and developed using aGloMax microplate reader.

FIGS. 16A and 16B. FIGS. 16A and 16B show that following incubation withz10, z10-MMAE, or z10-SN-38, MERTK is internalized by SKMel5 cells (FIG.16A) and MERTK is degraded (FIG. 16B). z10, z10-MMAE and z10-SN-38degrade MERTK to a similar extent. SKMel5 cells were incubated with 6.7nM of pHrodo-labeled z10, z10-MMAE or z10-SN-38. pHrodo signal is onlymeasurable in low pH conditions e.g. upon uptake into lysosomes. SurfaceMERTK was stained with a BV421-conjugated MERTK antibody. Binding wasmeasured by flow cytometry.

FIGS. 17A-17E. z10-MMAE and z10-SN-38 kill MERTK-expressing cancercells. SKMel5 (FIG. 17A) or RPMI8226 (FIG. 17B) cells were incubatedwith IgG control, IgG control conjugated to MMAE via a mc-mv-PABC linker(IgG-MMAE, see FIG. 13A where Ab is IgG) or z10-MMAE for 7 days. Cellviability was measured using CellTiterGlo. -SKMel5 (FIG. 17C) orRPMI8226 (FIG. 17D) cells were incubated with IgG control, IgG controlconjugated to SN-38 via a CL2A linker (IgG-SN-38, see FIG. 13B where Abis IgG and AA is lysine), or z10-SN-38 at indicated concentrations for 7days. Cell viability was measured using CellTiterGlo. FIG. 17E showsWestern Blot analysis of whole cell lysates prepared from SKMel5 orRPMI8226 cells.

FIGS. 18A and 18B. z10-MMAE inhibits lung colonization of MDA-MB-231-LM2TNBC cells. FIG. 18A shows lung colonization of MDA-MB-231-LM2 triplenegative breast cancer cells using IVIS imaging. FIG. 18B quantifies thelung colonization of MDA-MB-231-LM2 triple negative breast cancer cells.50,000 MDA-MB-231-LM2 TNBC cells were injected into the tail vein of NSGmice. Treatment started on the day of tumor cell inoculation and lungmetastatic colonization was monitored by bioluminescence imaging. Arrowsin FIG. 18B indicate days of dosing.** indicates a p-value of less than0.01.

FIGS. 19A and 19B. z10/z10-ADCs do not affect viability of MERTKexpressing M1 macrophages (FIG. 19A) or M2 macrophages (FIG. 19B). M2macrophages were differentiated by culturing 8×10⁴ monocytes in PBMCmedium with M-CSF (50 ng/mL) for 8 days. M1 macrophages weredifferentiated by culturing 8×10⁴ monocytes in PBMC medium with GM-CSF(20 ng/mL) for 5 days, then cultured with GM-CSF (20 ng/mL), IFNg (20ng/mL), IL-6 (20 ng/mL), and LPS (10 pg/mL) for additional 3 days. M2and M1 macrophages were treated with indicated antibodies or ADCs for 4days in differentiating medium and viability was assessed usingCellTiter-Glo 2.0 Cell Viability Assay.

5. DETAILED DESCRIPTION

Provided herein are anti-MERTK antibodies (e.g., monoclonal antibodies),and antigen-binding fragments thereof, that specifically bind to MERTK(e.g., human MERTK).

In certain embodiments, an anti-MERTK antibody (e.g., a humanizedantibody) or antigen-binding fragment thereof, described hereinspecifically recognizes the extracellular domain of MERTK (e.g., humanMERTK). In a particular embodiment, an anti-MERTK antibody (e.g., ahumanized antibody) or antigen-binding fragment thereof described hereindoes not bind human Axl, human Tyro 3, or murine MERTK as detected by atechnique known to one of skill in the art, or described herein. In aspecific embodiment, an anti-MERTK antibody (e.g., a humanized antibody)or antigen-binding fragment thereof described herein does not bind tohuman Axl, human Tyro3, or murine MERTK as assessed with an affinitybinding assay using ELISA such as described in Section 6.2, infra.

In a specific embodiment, an anti-MERTK antibody (e.g., a humanizedantibody) or antigen-binding fragment thereof described hereinspecifically binds to human MERTK protein comprising the amino acidsequence of SEQ ID NO: 131. In another specific embodiment, ananti-MERTK antibody (e.g., a humanized antibody) or antigen-bindingfragment thereof described herein specifically binds to theextracellular region of human MERTK, comprising the amino acid sequenceof SEQ ID NO: 132. In another specific embodiment, an anti-MERTKantibody (e.g., a humanized antibody) or antigen-binding fragmentthereof described herein specifically binds to SEQ ID NO: 132. In aspecific embodiment, an anti-MERTK antibody (e.g., a humanized antibody)or antigen-binding fragment thereof is bivalent. In a specificembodiment, an anti-MERTK antibody (e.g., a humanized antibody) orantigen-binding fragment thereof described herein comprises twoantigen-binding sites that bind to MERTK (e.g., human MERTK). In aparticular embodiment, the two antigen-binding sites bind to the sameepitope on MERTK (e.g., human MERTK). In a particular embodiment, thetwo-antigen binding sites comprise identical CDRs.

In certain embodiments, an anti-MERTK antibody (e.g., a humanizedantibody) or antigen-binding fragment thereof binds to a recombinantMERTK-Fc chimeric (e.g., a recombinant human MERTK-Fc chimeric) with aK_(D) of about 1 pM to 10 nM as determined using a technique known toone of skill in the art or described herein. In a specific embodiment,an anti-MERTK antibody (e.g., a humanized antibody) or anantigen-binding fragment thereof described herein binds to a recombinantMERTK-Fc chimeric (e.g., a recombinant human MERTK-Fc chimeric) with aK_(D) of about 1 pM to about 6 pM as determined using surface plasmonresonance (SPR), such as described in Section 6, infra. In certainembodiments, an anti-MERTK antibody (e.g., a humanized antibody) or anantigen-binding fragment thereof described herein binds to MERTK (e.g,.human MERTK) with an EC₅₀ of about 1 nM to 20 nM using an assaydescribed herein or known to one of skill in the art. In a specificcertain embodiment, an anti-MERTK antibody (e.g., a humanized antibody)or an antigen-binding fragment thereof as described herein binds toMERTK (e.g,. human MERTK) in an assay described in Section 6, infra,with a K_(D) of about 1 nM to 10 nM.

In certain embodiments, an anti-MERTK antibody (e.g., a humanizedantibody) or an antibody fragment thereof as described herein binds tohuman MERTK expressing cells (e.g., human MERTK cancer cells or M2macrophages) with an EC₅₀ of about 1 nM to 20 nM as assessed by flowcytometry such as described herein or known to one of skill in the art.In a specific embodiment, an anti-MERTK antibody (e.g., a humanizedantibody) or an antigen-binding fragment thereof as described hereinbinds to human MERTK expressing cells (e.g., human MERTK cancer cells orM2 macrophages) with an EC₅₀ of 1 nm to 10 nM as assessed by flowcytometry such as described in Section 6.2, infra.

In certain embodiments, an anti-MERTK antibody (e.g., a humanizedantibody) or antigen-binding fragment thereof described herein decreasesthe expression level of human MERTK on cancer cells as assessed by anassay described herein (e.g., in Section 6.2, infra) or an assay knownto one of skill in the art. In a particular embodiment, an anti-MERTKantibody (e.g., a humanized antibody) or antigen-binding fragmentthereof described herein decreases the expression level of human MERTKon the melanoma cell line SKMEL5 as assessed by an assay describedherein (e.g., in Section 6.2, infra) or an assay known to one of skillin the art. In certain embodiments, an anti-MERTK antibody (e.g., ahumanized antibody) or antigen-binding fragment thereof decreases theexpression level of human MERTK on human M2 macrophages as assessed byan assay described herein (e.g., in Section 6.2, infra) or an assayknown to one of skill in the art. In certain embodiments, an anti-MERTKantibody (e.g., a humanized antibody) or antigen-binding fragmentthereof decreases the expression level of human MERTK by internalizationof MERTK as assessed by an assay described herein (e.g., in Section 6.2,infra) or an assay known to one of skill in the art. In certainembodiments, an anti-MERTK antibody (e.g., a humanized antibody) orantigen-binding fragment thereof decreases the expression level of humanMERTK by internalization into lysosomes as assessed by an assaydescribed herein (e.g., in Section 6.2, infra) or an assay known to oneof skill in the art.

In certain embodiments, an anti-MERTK antibody (e.g., a humanizedantibody) or an antigen-binding fragment thereof described hereinreduces Gas 6-induced AKT phosphorylation in human MERTK expressingcells (e.g., cancer cells) as assessed by an assay described herein(e.g., Section 6.2, infra) or an assay known to one of skill in the art.In a specific embodiment, an anti-MERTK antibody (e.g., a humanizedantibody) or an antigen-binding fragment thereof described hereinprevents Gas-6-induced AKT phosphorylation in human MERTK expressingcancer cells, such as e.g., melanoma SKMEL5 cells, as assessed by anassay described herein (e.g., Section 6.2, infra) or an assay known toone of skill in the art. In certain embodiments an anti-MERTK antibody(e.g., a humanized antibody) or an antigen-binding fragment thereofdescribed herein reduces Gas-6-induced phosphorylation of human MERTK inhuman MERTK expressing cells as assessed by an assay known to one ofskill in the art. In a specific embodiment, an anti-MERTK antibody(e.g., a humanized antibody) or an antigen-binding fragment thereofdescribed herein blocks Gas-6-induced activation of MERTK in human MERTKexpressing cancer cells (e.g., melanoma cells, such as, e.g., SKMEL5) asassessed by an assay known to one of skill in the art.

In certain embodiments, an anti-MERTK antibody (e.g., a humanizedantibody) or antigen-binding fragment described herein, reduces thecolony formation ability of cancer cells as assessed by an assaydescribed herein (e.g., in Section 6.2, infra) or an assay known to oneof skill in the art. In certain embodiments, an anti-MERTK antibody(e.g., a humanized antibody) or antigen-binding fragment thereofdescribed herein inhibits colony formation of cancer cells (e.g.melanoma cells such as SKMEL5).

In certain embodiments, an anti-MERTK antibody (e.g., a humanizedantibody) or antigen-binding fragment thereof described herein inducescytokine responses in M2 macrophages, CD14+ monocytes, or both, such asprovided in FIG. 10, as assessed using an assay described herein orknown to one of skill in the art.

In specific embodiments, an anti-MERTK antibody (e.g., a humanizedantibody) or antigen-binding fragment thereof described herein isisolated.

Antibodies described herein may include monoclonal antibodies orpolyclonal antibodies. In a specific embodiment, an antibody may be animmunoglobulin, a tetrameric antibody comprising two heavy chain and twolight chain molecules, an antibody light chain monomer, an antibodyheavy chain monomer, an antibody light chain dimer, an antibody heavychain dimer, an antibody light chain-antibody heavy chain pair, a singledomain antibody, monovalent antibodies, a single chain antibody, asingle-chain Fv (scFv), a disulfide-linked Fv (scFv), and ananti-idiotypic (anti-Id) antibody (including, e.g., anti-anti-Idantibody).

In certain embodiments, an antibody described herein may bemultispecific or bispecific. In certain embodiments, an antibodydescribed herein is a bispecific monoclonal antibody. In certainembodiments, an antibody described herein is monovalent. In a specificembodiment, an antibody described herein is bivalent. In a particularembodiment, an antibody described herein binds to a human MERTK oncells. In some embodiments, an antibody described herein ismonospecific. In certain embodiments, an antibody described herein isrecombinantly produced. In some embodiments, an antibody describedherein is a synthetic antibody. In a specific embodiment, an antibodydescribed herein is a humanized antibody. In other embodiments, anantibody described herein may be a human antibody.

The antibodies described herein can be of any type (e.g., IgG, IgE, IgM,IgD, IgA or IgY), any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ orIgA₂), or any subclass (e.g., IgG_(2a) or IgG_(2b)) of immunoglobulinmolecules. In certain embodiments, the antibody described herein is anIgG antibody, or a class or subclass thereof. In a specific embodiment,an antibody (e.g., a humanized antibody) described herein is amonoclonal antibody. In a specific embodiment, an anti-MERTK antibodydescribed herein is an IgG antibody.

As used herein, the terms “antigen-binding fragment”, “antigen-bindingregion”, and similar terms refer to a portion of an antibody moleculewhich comprises the amino acid residues that confer on the antibodymolecule its specificity for the antigen (e.g., the complementaritydetermining regions (CDR)). The antigen-binding region can be derivedfrom any animal species, such as rodents (e.g., mouse, rat or hamster)and humans. By way of example, antigen-binding fragments include Fabfragments, F(ab′)₂ fragments, and antigen binding fragments of any ofthe antibodies described above. In a specific embodiment, anantigen-binding fragment of a humanized antibody comprises a variableheavy chain region, a variable light chain region or both of the z10,z11 or z13 antibody described herein.

As used herein, the terms “variable region” or “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, which differs extensively in sequence amongantibodies and is used in the binding and specificity of a particularantibody for its particular antigen. The variability in sequence isconcentrated in those regions called complementarity determining regions(CDRs) while the more highly conserved regions in the variable domainare called framework regions (FR). Without wishing to be bound by anyparticular mechanism or theory, it is believed that the CDRs of thelight and heavy chains are primarily responsible for the interaction andspecificity of the antibody with antigen.

CDRs are defined in various ways in the art, including the Kabat,Chothia, AbM, contact, IMGT, and Exemplary definitions. The Kabatdefinition is based on sequence variability and is the most commonlyused definition to predict CDR regions (Kabat, Elvin A. et al.,Sequences of Proteins of Immunological Interest. Bethesda: NationalInstitutes of Health, 1983). The Chothia definition is based on thelocation of the structural loop regions (Chothia et al., (1987) J MolBiol 196: 901-917). The AbM definition, a compromise between the Kabatand Chothia definitions, is an integral suite of programs for antibodystructure modeling produced by the Oxford Molecular Group(bioinf.org.uk/abs) (Martin A C R et al., (1989) PNAS 86: 9268-9272).The contact definition is based on an analysis of the available complexcrystal structures (bioinf.org.uk/abs) (see MacCallum R M et al., (1996)J Mol Biol 5: 732-745). The IMGT definition is from the IMGT (“IMGT®,the international ImMunoGeneTics information System® website imgt.org,founder and director: Marie-Paule Lefranc, Montpellier, France). TheExemplary definition is a combination of AbM and Kabat (Presta et al.,(1997) Cancer Res 57: 4593-4599).

In a specific embodiment, a humanized antibody or an antigen-bindingfragment thereof described herein comprises a variable heavy chainregion, a variable light chain region, or both, wherein the variableheavy chain region, variable light chain region or both are the variableheavy chain region, the variable heavy chain region, or both, of thez10, z11 or z12 antibody, which are provided in Table 11, 12 and 13,respectively. In another specific embodiment, a humanized antibody or anantigen-binding fragment described herein comprises a variable heavychain region, a variable light chain region, or both, wherein thevariable heavy chain comprises the amino acid sequence of the variableheavy chain region in Table 14, and the variable light chain regioncomprises the amino acid sequence of the variable light chain region inTable 11, 12 or 13, infra. In specific embodiments, the humanizedantibody is monoclonal. In some embodiments, the humanized antibody isan immunoglobulin, a tetrameric antibody comprising two heavy chain andtwo light chain molecules, an antibody light chain monomer, an antibodyheavy chain monomer, an antibody light chain dimer, and antibody heavychain dimer, an antibody light chain-antibody heavy chain pair, a singledomain antibody, a single chain antibody, single chain Fv, adisulfide-linked Fv or an anti-idotypic antibody. The humanized antibodymay be of any type (e.g. IgG, IgE, IgM, IgD, IgA, or IgY), any class(IgG₁, IgG₂, IgG₃, IgG₄, IgA, or IgA₂), or any subclass (e.g. IgG_(2a)or IgG_(2b)) of immunoglobulin molecules. In a specific embodiment, ahumanized antibody described herein is an IgG, or a class or subclassthereof.

The application not only contemplates antibodies and antigen-bindingfragments thereof that comprise the sequences disclosed herein (e.g.,CDRs, framework regions, variable regions) but also antibodies andantigen-binding fragments thereof, that consist or consist essentiallyof the sequences disclosed herein. For example, an antibody may consistof the light and heavy chains sequences of the z10, z11 or z13antibodies described herein.

In a specific embodiment, provided herein is a chimeric anti-MERTKantibody or a antigen-binding fragment thereof, which comprises avariable heavy chain region, a variable light chain region or both,wherein the variable light chain comprises the amino acid sequence ofthe variable light chain region in Table 15, infra, and the variableheavy chain comprises the amino acid sequence of the variable heavychain region in Table 15, infra. In specific embodiments, the chimericantibody is monoclonal. In some embodiments, the chimeric antibody is animmunoglobulin, a tetrameric antibody comprising two heavy chain and twolight chain molecules, an antibody light chain monomer, an antibodyheavy chain monomer, an antibody light chain dimer, and antibody heavychain dimer, an antibody light chain-antibody heavy chain pair, a singledomain antibody, a single chain antibody, single chain Fv, adisulfide-linked Fv or an anti-idotypic antibody. The chimeric antibodycan be of any type (e.g. IgG, IgE, IgM, IgD, IgA, or IgY), any class(IgG₁, IgG₂, IgG₃, IgG₄, IgA, or IgA₂), or any subclass (e.g. IgG_(2a)or IgG_(2b)) of immunoglobulin molecules. In certain embodiments, achimeric antibody described herein is an IgG antibody, or a class orsubclass thereof.

In another aspect, provided herein are multispecific antibodies andheteroconjugate antibodies. In a specific embodiment, provided herein isa bispecific antibody which comprises two different antigen bindingregions, wherein one of the binding regions binds MERTK (e.g., humanMERTK), and comprises a variable heavy chain region, a variable lightchain region or both, of an antibody described herein (e.g., the z10,z11, z13 or xAb antibody), and the other binding region binds to anantigen of interest. In some embodiments, a bispecific antibodycomprises two different antigen binding regions, wherein one of thebinding regions specifically binds MERTK (e.g., human MERTK) and theother binding region binds an antigen of interest, and wherein thebinding region that specifically binds to MERTK (e.g., human MERTK)comprises a variable heavy chain region and a variable light chainregion of the z10, z12 or z13 antibody described herein. In a specificembodiment, the antigen of interest is an immune cell receptor or atumor-associated antigen. See section 5.1.3, infra, regarding bispecificantibodies.

Also provided herein are heteroconjugate antibodies. In a specificembodiment a heteroconjugate antibody comprises two monoclonalantibodies with specificities for two different antigens, wherein one ofthe monoclonal antibodies comprises a variable heavy chain region and avariable heavy light chain region of an described herein (e.g., the z10,z11, z13 or xAb antibody).

Also provided are isolated nucleic acids (polynucleotides), such ascomplementary DNA (cDNA), encoding the anti-MERTK antibodies, andantigen-binding fragments thereof described herein. In certainembodiments, provided herein are polynucleotides comprising the nucleicacid sequence of the VH, the VL, or both of the z10, z11, z13 or xAbantibody set forth in Table 21, 22, 23 or 24. In some embodiments,provided herein are polynucleotides comprising the heavy chain, lightchain, or both of the z10, z11, z13 or xAb antibody as set forth inTable 25, 26, 27 or 28.

Further provided are vectors (e.g., expression vectors) and cells (e.g.,host cells) comprising nucleic acids (polynucleotides) encoding theanti-MERTK antibodies or antigen-binding fragments thereof describedherein. Also provided are methods of making such antibodies.

In another aspect, provided herein are antibody-drug conjugatescomprising: (a) an antibody moiety that is an anti-MERTK antibody or anantigen-binding fragment thereof described herein, which specificallybinds to MERTK (e.g., human MERTK) described herein; (b) one or moredrug moieties, each drug moiety being a cytotoxic agent; and (c)optionally a linker; wherein the cytotoxic agent is conjugated directlyto the antibody moiety or is conjugated to the antibody moiety via thelinker. The term “conjugated” as used in this disclosure shall meancovalently bound, which can be directly or via an intervening covalentlybound structure.

In the case where the cytotoxic agent is a peptide or protein, or wherethe cytotoxic agent and the linker (if there is one) are peptides orproteins, nucleic acids encoding the antibody-drug conjugates are alsoprovided. In other aspects, provided herein are methods of producing theantibody-drug conjugates that comprise (i) the anti-MERTK antibodies orantigen-binding fragments thereof, and (ii) cytotoxic agents conjugateddirectly to the anti-MERTK antibodies or antigen-binding fragments, orconjugated to the anti-MERTK antibodies or antigen-binding fragments vialinkers.

In another aspect, provided herein are compositions (e.g. pharmaceuticalcompositions) comprising an anti-MERTK antibody or antigen-bindingfragment thereof described herein. In a specific embodiment, apharmaceutical composition comprises an anti-MERTK antibody orantigen-binding fragment thereof described herein, one or morepharmaceutically acceptable carriers, excipients, or both, andoptionally one or more other therapeutic agents.

In another aspect, provided herein are compositions (e.g. pharmaceuticalcompositions) comprising an antibody-drug conjugate described herein. Ina specific embodiment, a pharmaceutical composition comprises anantibody-drug conjugate described herein, one or more pharmaceuticallyacceptable carriers, excipients, or both, and optionally one or moreother therapeutic agents.

In another aspect, provided herein are methods of treating cancer in asubject, comprising administering to the subject an effective amount ofan anti-MERTK antibody or antigen-binding fragment thereof describedherein or a composition thereof. In a specific embodiment, theanti-MERTK antibody or an antigen-binding fragment thereof is ahumanized antibody or an antigen-binding fragment described herein. Inother embodiments, the anti-MERTK antibody or an antigen-bindingfragment thereof is a bispecific antibody or a heteroconjugate antibodydescribed herein. In some embodiments, the anti-MERTK antibody orantigen-binding fragment thereof is a chimeric antibody orantigen-binding fragment thereof.

In another aspect, provided herein is a method of treating cancer in asubject comprising administering to the subject an effective amount ofantibody-drug conjugate described herein. In a specific embodiment,provided herein is a method of treating cancer in a subject comprisingadministering to the subject an effective amount of an antibody-drugconjugate that comprises (i) an anti-MERTK antibody or antigen-bindingfragment thereof, and (ii) cytotoxic agents conjugated directly to theanti-MERTK antibody or antigen-binding fragments thereof, or conjugatedto the anti-MERTK antibody or antigen-binding fragments thereof vialinkers.

5.1. Antibodies

5.1.1. Sequences and Variants

Provided in Tables 1 and 7 or 8, infra, are VH CDRs and VL CDRs,respectively, of an anti-MERTK antibody or an antigen-binding fragmentthereof as defined using different systems. Tables 2, 3, 4, 5 and 6infra, provide VH framework regions as defined using different systemswhich may be combined with the CDRs in Table 1. In a specificembodiment, the CDRs in Table 1 are combined with the consensus sequenceVH framework region 3, using one numbering system, along the VHframework region 1, 2 and 4, using the same numbering system, which areprovided in Tables 2, 3, 4 or 6. Tables 9 and 10, infra, provide VLframework regions as defined using different systems which may becombined with the CDRs in Table 7 or 8. Tables 11, 12, 13 and 15 providethe amino acid sequences of the VH and VL of particular antibodies.Table 14 provides the amino acid sequence of a VH that may be combinedwith the VL provided in Tables 11, 12, 13 or 15. Tables 16, 17, 18, and20 provide the heavy chain and light chain amino acid sequences ofparticular antibodies. Table 19 provides a heavy chain amino acidsequence that may be combined with the light chain amino acid sequencein Table 16, 17 or 18. Tables 21, 22, 23 and 24 provide the nucleic acidsequences encoding the variable regions of particular antibodies. Tables25, 26, 27 and 28 provide the nucleic acid sequences encoding the heavychain and light chain of particular antibodies. Tables 29 and 30 provideexemplary human MERTK amino acid sequences to which an anti-MERTKantibody or an antigen-binding fragment thereof described herein maybind.

In specific embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof provided herein comprises the VH CDR1 of the antibodyin Table 1 as defined by Kabat, Chothia, AbM, Contact, IMGT, orExemplary. In some embodiments, an anti-MERTK antibody orantigen-binding fragment thereof provided herein comprises the VH CDR2of the antibody in Tables 1 as defined by Kabat, Chothia, AbM, Contact,IMGT, or Exemplary. In some embodiments, an anti-MERTK antibody orantigen-binding fragment thereof provided herein comprises the VH CDR3of the antibody in Table 1 as defined by Kabat, Chothia, AbM, Contact,IMGT, or Exemplary. In some embodiments, an anti-MERTK antibody orantigen-binding fragment thereof provided herein comprises one, two orall three of VH CDRs of an antibody in Table 1 as determined by onenumbering system.

In specific embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof provided herein comprises the VL CDR1 of the antibodyin Table 7 as defined by Kabat, Chothia, AbM, Contact, IMGT, orExemplary. In some embodiments, an anti-MERTK antibody orantigen-binding fragment thereof provided herein comprises the VL CDR2of the antibody in Table 7 as defined by Kabat, Chothia, AbM, Contact,IMGT, or Exemplary. In some embodiments, an anti-MERTK antibody orantigen-binding fragment thereof provided herein comprises the VL CDR3of the antibody in Table 7 as defined by Kabat, Chothia, AbM, Contact,IMGT, or Exemplary. In some embodiments, an anti-MERTK antibody orantigen-binding fragment thereof provided herein comprises one, two orall three of VL CDRs of an antibody in Table 7 as determined by onenumbering system.

TABLE 1 Antibody z10/z11/z13/xAb VH CDR Amino Acid Sequences DefinitionsVH CDR1 VH CDR2 VH CDR3 Kabat NYGMN WINTYTGEPTYADDFKG KSTVVSRYFDV(SEQ ID NO: 1) (SEQ ID NO: 6) (SEQ ID NO: 11) Chothia GYTFTNY TYTGSTVVSRYFD (SEQ ID NO: 2) (SEQ ID NO: 7) (SEQ ID NO: 12) AbM GYTFTNYGMNWINTYTGEPT KSTVVSRYFDV (SEQ ID NO: 3) (SEQ ID NO: 8) (SEQ ID NO: 11)Contact TNYGMN WMGWINTYTGEPT ARKSTVVSRYFD (SEQ ID NO: 4) (SEQ ID NO: 9)(SEQ ID NO: 13) IMGT GYTFTNYG INTYTGEP ARKSTVVSRYFDV (SEQ ID NO: 5)(SEQ ID NO: 10) (SEQ ID NO: 14) Exemplary GYTFTNYGMN WINTYTGEPTYADDFKGKSTVVSRYFDV (SEQ ID NO: 3) (SEQ ID NO: 6) (SEQ ID NO: 11)

TABLE 2 Antibody z10 VH Framework Region Amino Acid SequencesDefinitions VH FR1 VH FR2 VH FR3 VH FR4 Kabat QVQLVQSGAE WVRQAPGQGLRVTFTTDTSTS WGQGTTVTVSS VKKPGASVKV EWMG TAYMELRSLRS (SEQ ID NO: 18)SCKASGYTFT (SEQ ID NO: 16) DDMAVYYCAR (SEQ ID NO: 15)  (SEQ ID NO: 17)Chothia QVQLVQSGAE GMNWVRQAP EPTYADDFKGR VWGQGTTVTVS VKKPGASVKVGQGLEWMGWI VTFTTDTSTST S SCKAS N AYMELRSLRSD (SEQ ID NO: 22)(SEQ ID NO: 19) (SEQ ID NO: 20) DMAVYYCARK (SEQ ID NO: 21) AbMQVQLVQSGAE WVRQAPGQGL YADDFKGRVT WGQGTTVTVSS VKKPGASVKV EWMG FTTDTSTSTAY(SEQ ID NO: 26) SCKAS (SEQ ID NO: 24) MELRSLRSDD (SEQ ID NO: 23)MAVYYCAR (SEQ ID NO: 25) Contact QVQLVQSGAE WVRQAPGQGL YADDFKGRVTVWGQGTTVTVS VKKPGASVKV E FTTDTSTSTAY S SCKASGYTF (SEQ ID NO: 28)MELRSLRSDD (SEQ ID NO: 30) (SEQ ID NO: 27) MAVYYC (SEQ ID NO: 29) IMGTQVQLVQSGAE MNWVRQAPG TYADDFKGRV WGQGTTVTVSS VKKPGASVKV QGLEWMGWTFTTDTSTSTA (SEQ ID NO: 34) SCKAS (SEQ ID NO: 32) YMELRSLRSDD(SEQ ID NO: 31) MAVYYC (SEQ ID NO: 33) Exemplary QVQLVQSGAE WVRQAPGQGLRVTFTTDTSTS WGQGTTVTVSS VKKPGASVKV EWMG TAYMELRSLRS (SEQ ID NO: 37)SCKAS (SEQ ID NO: 36) DDMAVYYCAR (SEQ ID NO: 35) (SEQ ID NO: 17)

TABLE 3 Antibody z11 VH Framework Region Amino Acid SequencesDefinitions VH FR1 VH FR2 VH FR3 VH FR4 Kabat QVQLVQSGAE WVRQAPGQGLRVTMTLDTSTS WGQGTTVTVSS VKKPGASVKV EWMG TAYMELRSLRS (SEQ ID NO: 18)SCKASGYTFT (SEQ ID NO: 16) DDMAVYYCAR (SEQ ID NO: 15) (SEQ ID NO: 38)Chothia QVQLVQSGAE GMNWVRQAP EPTYADDFKGR VWGQGTTVTVS VKKPGASVKVGQGLEWMGWI VTMTLDTSTST S SCKAS N AYMELRSLRSD (SEQ ID NO: 22)(SEQ ID NO: 19) (SEQ ID NO: 20) DMAVYYCARK (SEQ ID NO: 39) AbMQVQLVQSGAE WVRQAPGQGL YADDFKGRVT WGQGTTVTVSS VKKPGASVKV EWMG MTLDTSTSTAY(SEQ ID NO: 26) SCKAS (SEQ ID NO: 24) MELRSLRSDD (SEQ ID NO: 23)MAVYYCAR (SEQ ID NO: 40) Contact QVQLVQSGAE WVRQAPGQGL YADDFKGRVTVWGQGTTVTVS VKKPGASVKV E MTLDTSTSTAY S SCKASGYTF (SEQ ID NO: 28)MELRSLRSDD (SEQ ID NO: 30) (SEQ ID NO: 27) MAVYYC (SEQ ID NO: 41) IMGTQVQLVQSGAE MNWVRQAPG TYADDFKGRV WGQGTTVTVSS VKKPGASVKV QGLEWMGWTMTLDTSTSTA (SEQ ID NO: 34) SCKAS (SEQ ID NO: 32) YMELRSLRSDD(SEQ ID NO: 31) MAVYYC (SEQ ID NO: 42) Exemplary QVQLVQSGAE WVRQAPGQGLRVTMTLDTSTS WGQGTTVTVSS VKKPGASVKV EWMG TAYMELRSLRS (SEQ ID NO: 37)SCKAS (SEQ ID NO: 36) DDMAVYYCAR (SEQ ID NO: 35) (SEQ ID NO: 38)

TABLE 4 Antibody z13 VH Framework Region Amino Acid SequencesDefinitions VH FR1 VH FR2 VH FR3 VH FR4 Kabat QVQLVQSGAE WVRQAPGQGLRVTFTLDTSTS WGQGTTVTVSS VKKPGASVKV EWMG TAYMELRSLRS (SEQ ID NO: 18)SCKASGYTFT (SEQ ID NO: 16) DDMAVYYCAR (SEQ ID NO: 15) (SEQ ID NO: 43)Chothia QVQLVQSGAE GMNWVRQAP EPTYADDFKGR VWGQGTTVTVS VKKPGASVKVGQGLEWMGWI VTFTLDTSTST S SCKAS N AYMELRSLRSD (SEQ ID NO: 22)(SEQ ID NO: 19) (SEQ ID NO: 20) DMAVYYCARK (SEQ ID NO: 44) AbMQVQLVQSGAE WVRQAPGQGL YADDFKGRVT WGQGTTVTVSS VKKPGASVKV EWMG FTLDTSTSTAY(SEQ ID NO: 26) SCKAS (SEQ ID NO: 24) MELRSLRSDD (SEQ ID NO: 23)MAVYYCAR (SEQ ID NO: 45) Contact QVQLVQSGAE WVRQAPGQGL YADDFKGRVTVWGQGTTVTVS VKKPGASVKV E FTLDTSTSTAY S SCKASGYTF (SEQ ID NO: 28)MELRSLRSDD (SEQ ID NO: 30) (SEQ ID NO: 27) MAVYYC (SEQ ID NO: 46) IMGTQVQLVQSGAE MNWVRQAPG TYADDFKGRV WGQGTTVTVSS VKKPGASVKV QGLEWMGWTFTLDTSTSTA (SEQ ID NO: 34) SCKAS (SEQ ID NO: 32) YMELRSLRSDD(SEQ ID NO: 31) MAVYYC (SEQ ID NO: 47) Exemplary QVQLVQSGAE WVRQAPGQGLRVTFTLDTSTS WGQGTTVTVSS VKKPGASVKV EWMG TAYMELRSLRS (SEQ ID NO: 37)SCKAS (SEQ ID NO: 36) DDMAVYYCAR (SEQ ID NO: 35) (SEQ ID NO: 43)

TABLE 5 Antibody VH FR3 Consensus Amino Acid Sequences(X₁ is F or M; X₂ is T or L) Def- initions VH FR3 KabatRVTX₁TX₂DTSTSTAYMELRSLRSDDMAVYYCAR (SEQ ID NO: 48) ChothiaEPTYADDFKGRVTX₁TX₂DTSTSTAYMELRSLRSDDMAVY YCARK (SEQ ID NO: 49) AbMYADDFKGRVTX₁TX₂DTSTSTAYMELRSLRSDDMAVYYCA R (SEQ ID NO: 50) ContactYADDFKGRVTX₁TX₂DTSTSTAYMELRSLRSDDMAVYYC (SEQ ID NO: 51) IMGTTYADDFKGRVTX₁TX₂DTSTSTAYMELRSLRSDDMAVYYC (SEQ ID NO: 52) ExemplaryRVTX₁TX₂DTSTSTAYMELRSLRSDDMAVYYCAR (SEQ ID NO: 53)

TABLE 6 Antibody xAb VH Framework Region Amino Acid SequencesDefinitions VH FR1 VH FR2 VH FR3 VH FR4 Kabat QIQLVQSGPEL WVKQAPGKGLRFVFSLETSAST WGAGTTVTVSS KKPGETVKISC KWMG AYLQINNLKNE (SEQ ID NO: 57)KASGYTFT (SEQ ID NO: 55) DMATYFCAR (SEQ ID NO: 54) (SEQ ID NO: 56)Chothia QIQLVQSGPEL GMNWVKQAP EPTYADDFKGR VWGAGTTVTVS KKPGETVKISCGKGLKWMGWI FVFSLETSAST S KAS N AYLQINNLKNE (SEQ ID NO: 61)(SEQ ID NO: 58) (SEQ ID NO: 59) DMATYFCARK (SEQ ID NO: 60) AbMQIQLVQSGPEL WVKQAPGKGL YADDFKGRFV WGAGTTVTVSS KKPGETVKISC KWMGFSLETSASTAY (SEQ ID NO: 57) KAS (SEQ ID NO: 55) LQINNLKNED(SEQ ID NO: 58) MATYFCAR (SEQ ID NO: 62) Contact QIQLVQSGPEL WVKQAPGKGLYADDFKGRFV VWGAGTTVTVS KKPGETVKISC K FSLETSASTAY S KASGYTF(SEQ ID NO: 64) LQINNLKNED (SEQ ID NO: 61) (SEQ ID NO: 63) MATYFC(SEQ ID NO: 65) IMGT QIQLVQSGPEL MNWVKQAPG TYADDFKGRF WGAGTTVTVSSKKPGETVKISC KGLKWMGW VFSLETSASTA (SEQ ID NO: 57) KAS (SEQ ID NO: 66)YLQINNLKNED (SEQ ID NO: 58) MATYFC (SEQ ID NO: 67) Exemplary QIQLVQSGPELWVKQAPGKGL RFVFSLETSAST WGAGTTVTVSS KKPGETVKISC KWMG AYLQINNLKNE(SEQ ID NO: 57) KAS (SEQ ID NO: 55) DMATYFCAR (SEQ ID NO: 58)(SEQ ID NO: 56)

TABLE 7 Antibody z10/z11/z13 VL CDR Amino Acid Sequences DefinitionsVL CDR1 VL CDR2 VL CDR3 Kabat KASQDVGDAVT WASTRHT QQYRSYPLT(SEQ ID NO: 68) (SEQ ID NO: 69) (SEQ ID NO: 70) Chothia SQDVGDA WASYRSYPL (SEQ ID NO: 71) (SEQ ID NO: 72) (SEQ ID NO: 73) AbM KASQDVGDAVTWASTRHT QQYRSYPLT (SEQ ID NO: 68) (SEQ ID NO: 69) (SEQ ID NO: 70)Contact GDAVTWY LLIYWASTRH QQYRSYPL (SEQ ID NO: 74) (SEQ ID NO: 75)(SEQ ID NO: 76) IMGT QDVGDA WAS QQYRSYPLT (SEQ ID NO: 77)(SEQ ID NO: 72) (SEQ ID NO: 70) Exemplary KASQDVGDAVT WASTRHT QQYRSYPLT(SEQ ID NO: 68) (SEQ ID NO: 69) (SEQ ID NO: 70)

TABLE 8 Antibody xAb VL CDR Amino Acid Sequences Definitions VL CDR1VL CDR2 VL CDR3 Kabat KASQDVGDAVT WASTRHT QQYRSYPLT (SEQ ID NO: 68)(SEQ ID NO: 69) (SEQ ID NO: 70) Chothia SQDVGDA WAS YRSYPL(SEQ ID NO: 71) (SEQ ID NO: 72) (SEQ ID NO: 73) AbM KASQDVGDAVT WASTRHTQQYRSYPLT (SEQ ID NO: 68) (SEQ ID NO: 69) (SEQ ID NO: 70) ContactGDAVTWC LLIYWASTRH QQYRSYPL (SEQ ID NO: 78) (SEQ ID NO: 75)(SEQ ID NO: 76) IMGT QDVGDA WAS QQYRSYPLT (SEQ ID NO: 77)(SEQ ID NO: 72) (SEQ ID NO: 70) Exemplary KASQDVGDAVT WASTRHT QQYRSYPLT(SEQ ID NO: 68) (SEQ ID NO: 69) (SEQ ID NO: 70)

TABLE 9 Antibody z10/z11/z13 VL Framework Region Amino Acid SequencesDefinitions VL FR1 VL FR2 VL FR3 VL FR4 Kabat DIQMTQSPSFL WYQQKPGKAPGVPDRFSGSGS FGQGTKLEIK SASVGDRVTIT KLLIY GTDFTLTISSLQ (SEQ ID NO: 82) C(SEQ ID NO: 80) PEDFATYYC (SEQ ID NO: 79) (SEQ ID NO: 81) ChothiaDIQMTQSPSFL VTWYQQKPGK TRHTGVPDRFS TFGQGTKLEIK SASVGDRVTIT APKLLIYGSGSGTDFTLTI (SEQ ID NO: 86) CKA (SEQ ID NO: 84) SSLQPEDFATY(SEQ ID NO: 83) YCQQ (SEQ ID NO: 85) AbM DIQMTQSPSFL WYQQKPGKAPGVPDRFSGSGS FGQGTKLEIK SASVGDRVTIT KLLIY GTDFTLTISSLQ (SEQ ID NO: 82) C(SEQ ID NO: 80) PEDFATYYC (SEQ ID NO: 79) (SEQ ID NO: 81) ContactDIQMTQSPSFL QQKPGKAPK TGVPDRFSGSG TFGQGTKLEIK SASVGDRVTIT(SEQ ID NO: 88) SGTDFTLTISSL (SEQ ID NO: 86) CKASQDV QPEDFATYYC(SEQ ID NO: 87) (SEQ ID NO: 89) IMGT DIQMTQSPSFL VTWYQQKPGK TRHTGVPDRFSFGQGTKLEIK SASVGDRVTIT APKLLIY GSGSGTDFTLTI (SEQ ID NO: 82) CKAS(SEQ ID NO: 84) SSLQPEDFATY (SEQ ID NO: 90) YC (SEQ ID NO: 91) ExemplaryDIQMTQSPSFL WYQQKPGKAP GVPDRFSGSGS FGQGTKLEIK SASVGDRVTIT KLLIYGTDFTLTISSLQ (SEQ ID NO: 82) C (SEQ ID NO: 80) PEDFATYYC (SEQ ID NO: 79)(SEQ ID NO: 81)

TABLE 10 Antibody xAb VL Framework Region Amino Acid SequencesDefinitions VL FR1 VL FR2 VL FR3 VL FR4 Kabat DIVLTQSHKFM WCQQKPGQPPGVPDRFTGSGS FGAGTKLELK STSVGDRVSIT KLLIY GTDFTLTINNV (SEQ ID NO: 95) C(SEQ ID NO: 93) QSEDLADYFC (SEQ ID NO: 92) (SEQ ID NO: 94) ChothiaDIVLTQSHKFM VTWCQQKPGQ TRHTGVPDRFT TFGAGTKLELK STSVGDRVSIT PPKLLIYGSGSGTDFTLTI (SEQ ID NO: 99) CKA (SEQ ID NO: 97) NNVQSEDLAD(SEQ ID NO: 96) YFCQQ (SEQ ID NO: 98) AbM DIVLTQSHKFM WCQQKPGQPPGVPDRFTGSGS FGAGTKLELK STSVGDRVSIT KLLIY GTDFTLTINNV (SEQ ID NO: 95) C(SEQ ID NO: 93) QSEDLADYFC (SEQ ID NO: 92) (SEQ ID NO: 94) ContactDIVLTQSHKFM QQKPGQPPK TGVPDRFTGSG TFGAGTKLELK STSVGDRVSIT (SEQ ID NO:SGTDFTLTINN (SEQ ID NO: 99) CKASQDV 101) VQSEDLADYFC (SEQ ID NO:(SEQ ID NO: 100) 102) IMGT DIVLTQSHKFM VTWCQQKPGQ TRHTGVPDRFT FGAGTKLELKSTSVGDRVSIT PPKLLIY GSGSGTDFTLTI (SEQ ID NO: 95) CKAS (SEQ ID NO: 97)NNVQSEDLAD (SEQ ID NO: YFC 103) (SEQ ID NO: 104) Exemplary DIVLTQSHKFMWCQQKPGQPP GVPDRFTGSGS FGAGTKLELK STSVGDRVSIT KLLIY GTDFTLTINNV(SEQ ID NO: 95) C (SEQ ID NO: 93) QSEDLADYFC (SEQ ID NO: 92)(SEQ ID NO: 94)

TABLE 11 Antibody z10 Variable Region Amino Acid Sequences VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRVTFTTDTSTSTAYMELRSLRSDDMAVYYCARKSTVVSRYFDVWGQGTTVTVSS (SEQ ID NO: 105) VLDIQMTQSPSFLSASVGDRVTITCKASQDVGDAVTWYQQKPGKAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRSYPLTFGQGTKLEIK (SEQ ID NO: 106)

TABLE 12 Antibody z11 Variable Region Amino Acid Sequences VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRVTMTLDTSTSTAYMELRSLRSDDMAVYYCARKSTVVSRYFDVWGQGTTVTVSS (SEQ ID NO: 107) VLDIQMTQSPSFLSASVGDRVTITCKASQDVGDAVTWYQQKPGKAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRSYPLTFGQGTKLEIK (SEQ ID NO: 106)

TABLE 13 Antibody z13 Variable Region Amino Acid Sequences VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRVTFTLDTSTSTAYMELRSLRSDDMAVYYCARKSTVVSRYFDVWGQGTTVTVSS (SEQ ID NO: 108) VLDIQMTQSPSFLSASVGDRVTITCKASQDVGDAVTWYQQKPGKAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRSYPLTFGQGTKLEIK (SEQ ID NO: 106)

TABLE 14 Antibody Heavy Chain Variable Region ConsensusAmino Acid Sequence (X1 is F or M; X2 is T or L) VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRVTX₁TX₂DTSTSTAYMELRSLRSDDMAVYYCARKSTVVSRYFDVWGQGTTVTVSS (SEQ ID NO: 109)

TABLE 15 Antibody xAb Variable Region Amino Acid Sequences VHQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLETSASTAYLQINNLKNEDMATYFCARKSTVVSRYFDVWGAGTTVTVSS (SEQ ID NO: 110) VLDIVLTQSHKFMSTSVGDRVSITCKASQDVGDAVTWCQQKPGQPPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTINNVQSEDLADYFCQQYRSYPLTFGAGTKLELK (SEQ ID NO: 111)

TABLE 16 Antibody z10 Heavy Chain and Light Chain Amino Acid SequencesHeavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQ ChainGLEWMGWINTYTGEPTYADDFKGRVTFTTDTSTSTAYMELRSLRSDDMAVYYCARKSTVVSRYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK(SEQ ID NO: 112) Light DIQMTQSPSFLSASVGDRVTITCKASQDVGDAVTWYQQKPGKA ChainPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 113)

TABLE 17 Antibody z11 Heavy Chain and Light Chain Amino Acid SequencesHeavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQ ChainGLEWMGWINTYTGEPTYADDFKGRVTMTLDTSTSTAYMELRSLRSDDMAVYYCARKSTVVSRYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK(SEQ ID NO: 114) Light DIQMTQSPSFLSASVGDRVTITCKASQDVGDAVTWYQQKPGKA ChainPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 113)

TABLE 18 Antibody z13 Heavy Chain and Light Chain Amino Acid SequencesHeavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQ ChainGLEWMGWINTYTGEPTYADDFKGRVTFTLDTSTSTAYMELRSLRSDDMAVYYCARKSTVVSRYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK(SEQ ID NO: 115) Light DIQMTQSPSFLSASVGDRVTITCKASQDVGDAVTWYQQKPGKA ChainPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 113)

TABLE 19 Antibody Heavy Chain Consensus Amino AcidSequences (X₁ is F or M; X₂ is T or L) HeavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQ ChainGLEWMGWINTYTGEPTYADDFKGRVTX₁TX₂DTSTSTAYMELRSLRSDDMAVYYCARKSTVVSRYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 116)

TABLE 20 Antibody xAb Heavy Chain and Light Chain Amino Acid SequencesHeavy QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGK ChainGLKWMGWINTYTGEPTYADDFKGRFVFSLETSASTAYLQINNLKNEDMATYFCARKSTVVSRYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK(SEQ ID NO: 117) Light DIVLTQSHKFMSTSVGDRVSITCKASQDVGDAVTWCQQKPGQP ChainPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTINNVQSEDLADYFCQQYRSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 118)

TABLE 21 Antibody z10 Variable Region DNA Sequences VHcaggtgcagctggtgcagagcggcgctgaggtgaagaagcccggcgctagcgtgaaggtgagctgcaaggccagcggctacaccttcaccaactacggcatgaactgggtgaggcaggctcctggacagggcctggagtggatgggctggatcaacacatacaccggcgagcccacctacgccgacgacttcaagggcagggtgacctttaccaccgacaccagcaccagcaccgcctacatggagctgaggagcctgagaagcgacgacatggccgtgtactactgcgccagaaagagcaccgtggtgtccaggtacttcgacgtgtggggccagggcaccacagtgaccgtgagcagc (SEQ ID NO: 119) VLgacatccagatgacccagagccccagcttcctgtccgctagcgtgggcgacagggtgaccatcacctgcaaggccagccaggacgtgggcgatgccgtgacctggtatcagcagaagcccggcaaggcccccaagctgctgatctactgggccagcacaaggcacacaggcgtgcccgacagattcagcggcagcggaagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccagcagtacaggagctaccccctgaccttcggccagggcaccaagctggaaatcaag (SEQ ID NO: 120)

TABLE 22 Antibody z11 Variable Region DNA Sequences VHcaggtgcagctggtgcagagcggagccgaggtgaagaagcctggcgccagcgtgaaggtgagctgcaaggccagcggctacaccttcaccaactacggcatgaactgggtgaggcaggctcctggccagggactggagtggatgggctggatcaacacctacaccggcgagcccacctacgccgacgacttcaagggcagggtgaccatgaccctggacaccagcaccagcaccgcctacatggagctgaggagcctgaggagcgacgacatggccgtgtactactgcgccaggaagagcaccgtggtgtccaggtacttcgacgtgtggggacagggcaccaccgtgaccgtgagcagc (SEQ ID NO: 121) VLgacatccagatgacccagagccccagcttcctgtccgctagcgtgggcgacagggtgaccatcacctgcaaggccagccaggacgtgggcgatgccgtgacctggtatcagcagaagcccggcaaggcccccaagctgctgatctactgggccagcacaaggcacacaggcgtgcccgacagattcagcggcagcggaagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccagcagtacaggagctaccccctgaccttcggccagggcaccaagctggaaatcaag (SEQ ID NO: 120)

TABLE 23 Antibody z13 Variable Region DNA Sequences VHcaggtgcagctggtgcagagcggagccgaagtgaagaagcccggcgccagcgtgaaggtgagctgcaaggccagcggctacaccttcaccaactacggcatgaactgggtgagacaggcccctggacagggactggagtggatgggctggatcaacacctacaccggcgagcccacctacgccgacgacttcaagggcagggtgaccttcaccctggacaccagcaccagcaccgcctacatggagctgaggagcctgaggagcgacgacatggccgtgtactattgcgccaggaagagcaccgtggtgagcaggtacttcgacgtgtggggccagggaaccaccgtgaccgtgagcagc (SEQ ID NO: 122) VLgacatccagatgacccagagccccagcttcctgtccgctagcgtgggcgacagggtgaccatcacctgcaaggccagccaggacgtgggcgatgccgtgacctggtatcagcagaagcccggcaaggcccccaagctgctgatctactgggccagcacaaggcacacaggcgtgcccgacagattcagcggcagcggaagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccagcagtacaggagctaccccctgaccttcggccagggcaccaagctggaaatcaag (SEQ ID NO: 120)

TABLE 24 Antibody xAb Variable Region DNA Sequences VHcagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctggatataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagccaacatatgctgatgacttcaagggacggtttgtcttctctttggaaacctctgccagcactgcctacttgcagatcaacaacctcaaaaatgaggacatggccacatatttctgtgcaagaaaaagtacggtagtaagtaggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctca (SEQ ID NO: 123) VLgacattgtgctgacccagtctcacaaattcatgtccacatcagtaggagacagggtcagcatcacctgcaaggccagtcaggatgtgggtgatgctgtaacctggtgtcaacagaaaccaggtcaacctcctaaactactgatttactgggcatccacccggcacactggagtccctgatcgcttcacaggcagtgggtctgggacagatttcactctcaccattaacaatgtgcagtctgaggacttggcagattatttctgtcagcaatatcgcagctatcctctcacgttcggtgctgggaccaagctggagctgaaa (SEQ ID NO: 124)

TABLE 25 Antibody z10 Heavy Chain and Light Chain DNA Sequences Heavycaggtgcagctggtgcagagcggcgctgaggtgaagaagcccg Chaingcgctagcgtgaaggtgagctgcaaggccagcggctacaccttcaccaactacggcatgaactgggtgaggcaggctcctggacagggcctggagtggatgggctggatcaacacatacaccggcgagcccacctacgccgacgacttcaagggcagggtgacctttaccaccgacaccagcaccagcaccgcctacatggagctgaggagcctgagaagcgacgacatggccgtgtactactgcgccagaaagagcaccgtggtgtccaggtacttcgacgtgtggggccagggcaccacagtgaccgtgagcagcgcgtcgaccaagggcccatccgtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcctggaactcaggcgctctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtctacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagataag cctgtctccgggtaaatga(SEQ ID NO: 125) Light gacatccagatgacccagagccccagcttcctgtccgctagcg Chaintgggcgacagggtgaccatcacctgcaaggccagccaggacgtgggcgatgccgtgacctggtatcagcagaagcccggcaaggcccccaagctgctgatctactgggccagcacaaggcacacaggcgtgcccgacagattcagcggcagcggaagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccagcagtacaggagctaccccctgaccttcggccagggcaccaagctggaaatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttga (SEQ ID NO: 126)

TABLE 26 Antibody z11 Heavy Chain and Light Chain DNA Sequences Heavycaggtgcagctggtgcagagcggagccgaggtgaagaagcctg Chaingcgccagcgtgaaggtgagctgcaaggccagcggctacaccttcaccaactacggcatgaactgggtgaggcaggctcctggccagggactggagtggatgggctggatcaacacctacaccggcgagcccacctacgccgacgacttcaagggcagggtgaccatgaccctggacaccagcaccagcaccgcctacatggagctgaggagcctgaggagcgacgacatggccgtgtactactgcgccaggaagagcaccgtggtgtccaggtacttcgacgtgtggggacagggcaccaccgtgaccgtgagcagcgcgtcgaccaagggcccatccgtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcctggaactcaggcgctctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtctacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcttaa gcctgtctccgggtaaatga(SEQ ID NO: 127) Light gacatccagatgacccagagccccagcttcctgtccgctagcg Chaintgggcgacagggtgaccatcacctgcaaggccagccaggacgtgggcgatgccgtgacctggtatcagcagaagcccggcaaggcccccaagctgctgatctactgggccagcacaaggcacacaggcgtgcccgacagattcagcggcagcggaagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccagcagtacaggagctaccccctgaccttcggccagggcaccaagctggaaatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttga (SEQ ID NO: 126)

TABLE 27 Antibody z13 Heavy Chain and Light Chain DNA Sequences Heavycaggtgcagctggtgcagagcggagccgaagtgaagaagcccg Chaingcgccagcgtgaaggtgagctgcaaggccagcggctacaccttcaccaactacggcatgaactgggtgagacaggcccctggacagggactggagtggatgggctggatcaacacctacaccggcgagcccacctacgccgacgacttcaagggcagggtgaccttcaccctggacaccagcaccagcaccgcctacatggagctgaggagcctgaggagcgacgacatggccgtgtactattgcgccaggaagagcaccgtggtgagcaggtacttcgacgtgtggggccagggaaccaccgtgaccgtgagcagcgcgtcgaccaagggcccatccgtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcctggaactcaggcgctctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtctacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcttaa gcctgtctccgggtaaatga(SEQ ID NO: 128) Light gacatccagatgacccagagccccagcttcctgtccgctagcg Chaintgggcgacagggtgaccatcacctgcaaggccagccaggacgtgggcgatgccgtgacctggtatcagcagaagcccggcaaggcccccaagctgctgatctactgggccagcacaaggcacacaggcgtgcccgacagattcagcggcagcggaagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccagcagtacaggagctaccccctgaccttcggccagggcaccaagctggaaatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttga (SEQ ID NO: 126)

TABLE 28 Antibody xAb Heavy Chain and Light Chain DNA Sequences Heavycagatccagttggtgcagtctggacctgagctgaagaagcctg Chaingagagacagtcaagatctcctgcaaggcttctggatataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagccaacatatgctgatgacttcaagggacggtttgtcttctctttggaaacctctgccagcactgcctacttgcagatcaacaacctcaaaaatgaggacatggccacatatttctgtgcaagaaaaagtacggtagtaagtaggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctcagcgtcgaccaagggcccatccgtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcctggaactcaggcgctctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtctacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcttaa gcctgtctccgggtaaatga(SEQ ID NO: 129) Light gacattgtgctgacccagtctcacaaattcatgtccacatcag Chaintaggagacagggtcagcatcacctgcaaggccagtcaggatgtgggtgatgctgtaacctggtgtcaacagaaaccaggtcaacctcctaaactactgatttactgggcatccacccggcacactggagtccctgatcgcttcacaggcagtgggtctgggacagatttcactctcaccattaacaatgtgcagtctgaggacttggcagattatttctgtcagcaatatcgcagctatcctctcacgttcggtgctgggaccaagctggagctgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttga (SEQ ID NO: 130)

TABLE 29 MERTK Protein Sequences Full-MGPAPLPLLLGLFLPALWRRAITEAREEAKPYPLFPGPF LengthPGSLQTDHTPLLSLPHASGYQPALMFSPTQPGRPHTGNV HumanAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSI MERTKSVPNIYQDTTISWWKDGKELLGAHHAITQFYPDDEVTAI (Swiss-IASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQG ProtLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQN ID:SSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVS Q12866.2)KGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAPSSTPAPGNADPVLIIFGCFCGFILIGLILYISLAIRKRVQETKFGNAFTEEDSELVVNYIAKKSFCRRAIELTLHSLGVSEELQNKLEDVVIDRNLLILGKILGEGEFGSVMEGNLKQEDGTSLKVAVKTMKLDNSSQREIEEFLSEAACMKDFSHPNVIRLLGVCIEMSSQGIPKPMVILPFMKYGDLHTYLLYSRLETGPKHIPLQTLLKFMVDIALGMEYLSNRNFLHRDLAARNCMLRDDMTVCVADFGLSKKIYSGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWAFGVTMWEIATRGMTPYPGVQNHEMYDYLLHGHRLKQPEDCLDELYEIMYSCWRTDPLDRPTFSVLRLQLEKLLESLPDVRNQADVIYVNTQLLESSEGLAQGSTLAPLDLNIDPDSIIASCTPRAAISVVTAEVHDSKPHEGRYILNGGSEEWEDLTSAPSAAVTAEKNSVLPGERLVRNGVSWSHSSML PLGSSLPDELLFADDSSEGSEVLM(SEQ ID NO: 131) Extra- REEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPALM cellularFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHI Doman ofILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGAHH MERTKAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINN UsedEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFNLTCQA forVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAV Immuni-FSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAH zationSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAPSST PAPGNA (SEQ ID NO: 132)

In a specific embodiment, provided herein is an anti-MERTK antibody oran antigen-binding fragment thereof that specifically binds to MERTK(e.g., human MERTK), wherein the antibody or antigen-binding fragmentthereof comprises a framework region (FR) 1, a variable heavy chainregion (VH) CDR1, an FR2, a VH CDR2, an FR3, a VH CDR3 and an FR4, andwherein the VH CDR1, VH CDR2, VH CDR3 comprise the amino acid sequencesof the VH CDR1, VH CDR2, and VH CDR3 set forth in Table 1 using theExemplary numbering system, and FR1, FR2, FR3 and FR4 comprise the aminoacid sequences of the FR1, FR2, FR3, and FR4 set forth in Table 2, 3, 4or 5 using the Exemplary numbering system. In another specificembodiment, provided herein is an anti-MERTK or an antigen-bindingfragment thereof that specifically binds to MERTK (e.g., human MERTK),wherein the antibody or antigen-binding fragment thereof comprises aframework region (FR) 1, a variable heavy chain region (VH) CDR1, anFR2, a VH CDR2, an FR3, a VH CDR3 and an FR4, and wherein the VH CDR1,VH CDR2, VH CDR3 comprise the amino acid sequences of the VH CDR1, VHCDR2, and VH CDR3 set forth in Table 1 using the Kabat numbering system,and FR1, FR2, FR3 and FR4 comprise the amino acid sequences of the FR1,FR2, FR3, and FR4 set forth in Table 2, 3, 4 or 5 using the Kabatnumbering system. In another specific embodiment, provided herein is ananti-MERTK antibody or an antigen-binding fragment thereof thatspecifically binds to MERTK (e.g., human MERTK), wherein the antibody orantigen-binding fragment thereof comprises a framework region (FR) 1, avariable heavy chain region (VH) CDR1, an FR2, a VH CDR2, an FR3, a VHCDR3 and an FR4, and wherein the VH CDR1, VH CDR2, VH CDR3 comprise theamino acid sequences of the VH CDR1, VH CDR2, and VH CDR3 set forth inTable 1 using the Chothia numbering system, and FR1, FR2, FR3 and FR4comprise the amino acid sequences of the FR1, FR2, FR3, and FR4 setforth in Table 2, 3, 4 or 5 using the Chothia numbering system. Inanother specific embodiment, provided herein is an anti-MERTK antibodyor an antigen-binding fragment thereof that specifically binds to MERTK(e.g., human MERTK), wherein the antibody or antigen-binding fragmentthereof comprises a framework region (FR) 1, a variable heavy chainregion (VH) CDR1, an FR2, a VH CDR2, an FR3, a VH CDR3 and an FR4, andwherein the VH CDR1, VH CDR2, VH CDR3 comprise the amino acid sequencesof the VH CDR1, VH CDR2, and VH CDR3 set forth in Table 1 using the AbMnumbering system, and FR1, FR2, FR3 and FR4 comprise the amino acidsequences of the FR1, FR2, FR3, and FR4 set forth in Table 2, 3, 4 or 5using the AbM numbering system. In another specific embodiment, providedherein is an anti-MERTK antibody or an antigen-binding fragment thereofthat specifically binds to MERTK (e.g., human MERTK), wherein theantibody or antigen-binding fragment thereof comprises a frameworkregion (FR) 1, a variable heavy chain region (VH) CDR1, an FR2, a VHCDR2, an FR3, a VH CDR3 and an FR4, and wherein the VH CDR1, VH CDR2, VHCDR3 comprise the amino acid sequences of the VH CDR1, VH CDR2, and VHCDR3 set forth in Table 1 using the Contact numbering system, and FR1,FR2, FR3 and FR4 comprise the amino acid sequences of the FR1, FR2, FR3,and FR4 set forth in Table 2, 3, 4 or 5 using the Contact numberingsystem. In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a framework region (FR) 1, a variable heavychain region (VH) CDR1, an FR2, a VH CDR2, an FR3, a VH CDR3 and an FR4,and wherein the VH CDR1, VH CDR2, VH CDR3 comprise the amino acidsequences of the VH CDR1, VH CDR2, and VH CDR3 set forth in Table 1using the IMGT numbering system, and FR1, FR2, FR3 and FR4 comprise theamino acid sequences of the FR1, FR2, FR3, and FR4 set forth in Table 2,3, 4 or 5 using the IMGT numbering system.

In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a framework region (FR) 1, a variable heavychain region (VH) CDR1, an FR2, a VH CDR2, an FR3, a VH CDR3 and an FR4,and wherein the VH CDR1, VH CDR2, VH CDR3 comprise the amino acidsequences of the VH CDR1, VH CDR2, and VH CDR3 set forth in Table 1using the one numbering system, and FR1, FR2, and FR4 comprise the aminoacid sequences of the FR1, FR2, and FR4 set forth in Table 2, 3, or 4using the same numbering system, and the FR3 comprises the amino acidsequence of the FR3 set forth in Table 5 using the same numberingsystem.

In a specific embodiment, provided herein is an anti-MERTK antibody oran antigen-binding fragment thereof that specifically binds to MERTK(e.g., human MERTK), wherein the antibody or antigen-binding fragmentthereof comprises a variable heavy chain region (VH), wherein the VHcomprises a framework region (FR) 1, a VH CDR1, an FR2, a VH CDR2, anFR3, a VH CDR3 and an FR4, and wherein the VH CDR1, VH CDR2, VH CDR3comprise the amino acid sequences of the VH CDR1, VH CDR2, and VH CDR3set forth in Table 1 using the Exemplary numbering system, and FR1, FR2,FR3 and FR4 comprise the amino acid sequences of the FR1, FR2, FR3, andFR4 set forth in Table 2, 3, 4 or 5 using the Exemplary numberingsystem. In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH), whereinthe VH comprises a framework region (FR) 1, a VH CDR1, an FR2, a VHCDR2, an FR3, a VH CDR3 and an FR4, and wherein the VH CDR1, VH CDR2, VHCDR3 comprise the amino acid sequences of the VH CDR1, VH CDR2, and VHCDR3 set forth in Table 1 using the Kabat numbering system, and FR1,FR2, FR3 and FR4 comprise the amino acid sequences of the FR1, FR2, FR3,and FR4 set forth in Table 2, 3, 4 or 5 using the Kabat numberingsystem. In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH), whereinthe VH comprises a framework region (FR) 1, a VH CDR1, an FR2, a VHCDR2, an FR3, a VH CDR3 and an FR4, and wherein the VH CDR1, VH CDR2, VHCDR3 comprise the amino acid sequences of the VH CDR1, VH CDR2, and VHCDR3 set forth in Table 1 using the Chothia numbering system, and FR1,FR2, FR3 and FR4 comprise the amino acid sequences of the FR1, FR2, FR3,and FR4 set forth in Table 2, 3, 4 or 5 using the Chothia numberingsystem. In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH), whereinthe VH comprises a framework region (FR) 1, a VH CDR1, an FR2, a VHCDR2, an FR3, a VH CDR3 and an FR4, and wherein the VH CDR1, VH CDR2, VHCDR3 comprise the amino acid sequences of the VH CDR1, VH CDR2, and VHCDR3 set forth in Table 1 using the AbM numbering system, and FR1, FR2,FR3 and FR4 comprise the amino acid sequences of the FR1, FR2, FR3, andFR4 set forth in Table 2, 3, 4 or 5 using the AbM numbering system. Inanother specific embodiment, provided herein is an anti-MERTK antibodyor an antigen-binding fragment thereof that specifically binds to MERTK(e.g., human MERTK), wherein the antibody or antigen-binding fragmentthereof comprises a variable heavy chain region (VH), wherein the VHcomprises a framework region (FR) 1, a VH CDR1, an FR2, a VH CDR2, anFR3, a VH CDR3 and an FR4, and wherein the VH CDR1, VH CDR2, VH CDR3comprise the amino acid sequences of the VH CDR1, VH CDR2, and VH CDR3set forth in Table 1 using the Contact numbering system, and FR1, FR2,FR3 and FR4 comprise the amino acid sequences of the FR1, FR2, FR3, andFR4 set forth in Table 2, 3, 4 or 5 using the Contact numbering system.In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH), whereinthe VH comprises a framework region (FR) 1, a VH CDR1, an FR2, a VHCDR2, an FR3, a VH CDR3 and an FR4, and wherein the VH CDR1, VH CDR2, VHCDR3 comprise the amino acid sequences of the VH CDR1, VH CDR2, and VHCDR3 set forth in Table 1 using the IMGT numbering system, and FR1, FR2,FR3 and FR4 comprise the amino acid sequences of the FR1, FR2, FR3, andFR4 set forth in Table 2, 3, 4 or 5 using the IMGT numbering system.

In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH), whereinthe VH comprises a framework region (FR) 1, a VH CDR1, an FR2, a VHCDR2, an FR3, a VH CDR3 and an FR4, and wherein the VH CDR1, VH CDR2, VHCDR3 comprise the amino acid sequences of the VH CDR1, VH CDR2, and VHCDR3 set forth in Table 1 using one numbering system, and FR1, FR2, andFR4 comprise the amino acid sequences of the FR1, FR2, and FR4 set forthin Table 2, 3, or 4 using the same numbering system, and the FR3comprises the amino acid sequence of the FR3 set forth in Table 5 usingthe same numbering system.

In a specific embodiment, provided herein is an anti-MERTK antibody oran antigen-binding fragment thereof that specifically binds to MERTK(e.g., human MERTK), wherein the antibody or antigen-binding fragmentthereof comprises a framework region (FR) 1, a variable light chainregion (VL) CDR1, an FR2, a VL CDR2, an FR3, a VL CDR3 and an FR4, andwherein the VL CDR1, VL CDR2, VL CDR3 comprise the amino acid sequencesof the VL CDR1, VL CDR2, and VL CDR3 set forth in Table 7 using theExemplary numbering system, and FR1, FR2, FR3 and FR4 comprise the aminoacid sequences of the FR1, FR2, FR3, and FR4 set forth in Table 9 usingthe Exemplary numbering system. In another specific embodiment, providedherein is an anti-MERTK antibody or an antigen-binding fragment thereofthat specifically binds to MERTK (e.g., human MERTK), wherein theantibody or antigen-binding fragment thereof comprises a frameworkregion (FR) 1, a variable light chain region (VL) CDR1, an FR2, a VLCDR2, an FR3, a VL CDR3 and an FR4, and wherein the VL CDR1, VL CDR2, VLCDR3 comprise the amino acid sequences of the VL CDR1, VL CDR2, and VLCDR3 set forth in Table 7 using the Kabat numbering system, and FR1,FR2, FR3 and FR4 comprise the amino acid sequences of the FR1, FR2, FR3,and FR4 set forth in Table 9 using the Kabat numbering system. Inanother specific embodiment, provided herein is an anti-MERTK antibodyor an antigen-binding fragment thereof that specifically binds to MERTK(e.g., human MERTK), wherein the antibody or antigen-binding fragmentthereof comprises a framework region (FR) 1, a variable light chainregion (VL) CDR1, an FR2, a VL CDR2, an FR3, a VL CDR3 and an FR4, andwherein the VL CDR1, VL CDR2, VL CDR3 comprise the amino acid sequencesof the VL CDR1, VL CDR2, and VL CDR3 set forth in Table 7 using theChothia numbering system, and FR1, FR2, FR3 and FR4 comprise the aminoacid sequences of the FR1, FR2, FR3, and FR4 set forth in Table 9 usingthe Chothia numbering system. In another specific embodiment, providedherein is an anti-MERTK antibody or an antigen-binding fragment thereofthat specifically binds to MERTK (e.g., human MERTK), wherein theantibody or antigen-binding fragment thereof comprises a frameworkregion (FR) 1, a variable light chain region (VL) CDR1, an FR2, a VLCDR2, an FR3, a VL CDR3 and an FR4, and wherein the VL CDR1, VL CDR2, VLCDR3 comprise the amino acid sequences of the VL CDR1, VL CDR2, and VLCDR3 set forth in Table 7 using the AbM numbering system, and FR1, FR2,FR3 and FR4 comprise the amino acid sequences of the FR1, FR2, FR3, andFR4 set forth in Table 9 using the AbM numbering system. In anotherspecific embodiment, provided herein is an anti-MERTK antibody or anantigen-binding fragment thereof that specifically binds to MERTK (e.g.,human MERTK), wherein the antibody or antigen-binding fragment thereofcomprises a framework region (FR) 1, a variable light chain region (VL)CDR1, an FR2, a VL CDR2, an FR3, a VL CDR3 and an FR4, and wherein theVL CDR1, VL CDR2, VL CDR3 comprise the amino acid sequences of the VLCDR1, VL CDR2, and VL CDR3 set forth in Table 7 using the Contactnumbering system, and FR1, FR2, FR3 and FR4 comprise the amino acidsequences of the FR1, FR2, FR3, and FR4 set forth in Table 9 using theContact numbering system. In another specific embodiment, providedherein is an anti-MERTK antibody or an antigen-binding fragment thereofthat specifically binds to MERTK (e.g., human MERTK), wherein theantibody or antigen-binding fragment thereof comprises a frameworkregion (FR) 1, a variable light chain region (VL) CDR1, an FR2, a VLCDR2, an FR3, a VL CDR3 and an FR4, and wherein the VL CDR1, VL CDR2, VLCDR3 comprise the amino acid sequences of the VL CDR1, VL CDR2, and VLCDR3 set forth in Table 7 using the IMGT numbering system, and FR1, FR2,FR3 and FR4 comprise the amino acid sequences of the FR1, FR2, FR3, andFR4 set forth in Table 9 using the IMGT numbering system.

In a specific embodiment, provided herein is an anti-MERTK antibody oran antigen-binding fragment thereof that specifically binds to MERTK(e.g., human MERTK), wherein the antibody or antigen-binding fragmentthereof comprises a variable light chain region (VL), wherein the VLcomprises a framework region (FR) 1, a variable light chain region (VL)CDR1, an FR2, a VL CDR2, an FR3, a VL CDR3 and an FR4, and wherein theVL CDR1, VL CDR2, VL CDR3 comprise the amino acid sequences of the VLCDR1, VL CDR2, and VL CDR3 set forth in Table 7 using the Exemplarynumbering system, and FR1, FR2, FR3 and FR4 comprise the amino acidsequences of the FR1, FR2, FR3, and FR4 set forth in Table 9 using theExemplary numbering system. In another specific embodiment, providedherein is an anti-MERTK antibody or an antigen-binding fragment thereofthat specifically binds to MERTK (e.g., human MERTK), wherein theantibody or antigen-binding fragment thereof comprises a variable lightchain region (VL), wherein the VL comprises a framework region (FR) 1, avariable light chain region (VL) CDR1, an FR2, a VL CDR2, an FR3, a VLCDR3 and an FR4, and wherein the VL CDR1, VL CDR2, VL CDR3 comprise theamino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 set forth inTable 7 using the Kabat numbering system, and FR1, FR2, FR3 and FR4comprise the amino acid sequences of the FR1, FR2, FR3, and FR4 setforth in Table 9 using the Kabat numbering system. In another specificembodiment, provided herein is an anti-MERTK antibody or anantigen-binding fragment thereof that specifically binds to MERTK (e.g.,human MERTK), wherein the antibody or antigen-binding fragment thereofcomprises a variable light chain region (VL), wherein the VL comprises aframework region (FR) 1, a variable light chain region (VL) CDR1, anFR2, a VL CDR2, an FR3, a VL CDR3 and an FR4, and wherein the VL CDR1,VL CDR2, VL CDR3 comprise the amino acid sequences of the VL CDR1, VLCDR2, and VL CDR3 set forth in Table 7 using the Chothia numberingsystem, and FR1, FR2, FR3 and FR4 comprise the amino acid sequences ofthe FR1, FR2, FR3, and FR4 set forth in Table 9 using the Chothianumbering system. In another specific embodiment, provided herein is ananti-MERTK antibody or an antigen-binding fragment thereof thatspecifically binds to MERTK (e.g., human MERTK), wherein the antibody orantigen-binding fragment thereof comprises a variable light chain region(VL), wherein the VL comprises a framework region (FR) 1, a variablelight chain region (VL) CDR1, an FR2, a VL CDR2, an FR3, a VL CDR3 andan FR4, and wherein the VL CDR1, VL CDR2, VL CDR3 comprise the aminoacid sequences of the VL CDR1, VL CDR2, and VL CDR3 set forth in Table 7using the AbM numbering system, and FR1, FR2, FR3 and FR4 comprise theamino acid sequences of the FR1, FR2, FR3, and FR4 set forth in Table 9using the AbM numbering system. In another specific embodiment, providedherein is an anti-MERTK antibody or an antigen-binding fragment thereofthat specifically binds to MERTK (e.g., human MERTK), wherein theantibody or antigen-binding fragment thereof comprises a variable lightchain region (VL), wherein the VL comprises a framework region (FR) 1, avariable light chain region (VL) CDR1, an FR2, a VL CDR2, an FR3, a VLCDR3 and an FR4, and wherein the VL CDR1, VL CDR2, VL CDR3 comprise theamino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 set forth inTable 7 using the Contact numbering system, and FR1, FR2, FR3 and FR4comprise the amino acid sequences of the FR1, FR2, FR3, and FR4 setforth in Table 9 using the Contact numbering system. In another specificembodiment, provided herein is an anti-MERTK antibody or anantigen-binding fragment thereof that specifically binds to MERTK (e.g.,human MERTK), wherein the antibody or antigen-binding fragment thereofcomprises a variable light chain region (VL), wherein the VL comprises aframework region (FR) 1, a variable light chain region (VL) CDR1, anFR2, a VL CDR2, an FR3, a VL CDR3 and an FR4, and wherein the VL CDR1,VL CDR2, VL CDR3 comprise the amino acid sequences of the VL CDR1, VLCDR2, and VL CDR3 set forth in Table 7 using the IMGT numbering system,and FR1, FR2, FR3 and FR4 comprise the amino acid sequences of the FR1,FR2, FR3, and FR4 set forth in Table 9 using the IGMT numbering system.

In a specific embodiment, provided herein is an anti-MERTK antibody oran antigen-binding fragment thereof that specifically binds to MERTK(e.g., human MERTK), wherein the antibody or antigen-binding fragmentthereof comprises a variable heavy chain region (VH) and a variablelight chain region (VL), wherein the VH comprises a VH framework region(FR) 1, a VH CDR1, a VH FR2, a VH CDR2, a VH FR3, a VH CDR3 and a VHFR4, wherein the VL comprises a VL FR1, a VL CDR1, a VL FR2, a VL CDR2,a VL FR3, a VL CDR3, and a VL FR4, wherein the VH CDR1, VH CDR2, VH CDR3comprise the amino acid sequences of the VH CDR1, VH CDR2, and VH CDR3set forth in Table 1 using the Exemplary numbering system, wherein theVH FR1, VH FR2, VH FR3 and VH FR4 comprise the amino acid sequences ofthe VH FR1, VH FR2, VH FR3, and VH FR4 set forth in Table 2, 3, 4 or 5using the exemplary numbering system, wherein the VL CDR1, VL CDR2, andVL CDR3 comprise the amino acid sequences of the VL CDR1, VL CDR2, andVL CDR3 set forth in Table 7 using the Exemplary numbering system, andwherein the VL FR1, VL FR2, VL FR3, and VL FR4 comprise the amino acidsequences of the VL FR1, VL FR2, VL FR3, and VL FR4 set forth in Table 9using the exemplary numbering system. In another specific embodiment,provided herein is an anti-MERTK antibody or an antigen-binding fragmentthereof that specifically binds to MERTK (e.g., human MERTK), whereinthe antibody or antigen-binding fragment thereof comprises a variableheavy chain region (VH) and a variable light chain region (VL), whereinthe VH comprises a VH framework region (FR) 1, a VH CDR1, a VH FR2, a VHCDR2, a VH FR3, a VH CDR3 and a VH FR4, wherein the VL comprises a VLFR1, a VL CDR1, a VL FR2, a VL CDR2, a VL FR3, a VL CDR3, and a VL FR4,wherein the VH CDR1, VH CDR2, VH CDR3 comprise the amino acid sequencesof the VH CDR1, VH CDR2, and VH CDR3 set forth in Table 1 using theKabat numbering system, wherein the VH FR1, VH FR2, VH FR3 and VH FR4comprise the amino acid sequences of the VH FR1, VH FR2, VH FR3, and VHFR4 set forth in Table 2, 3, 4 or 5 using the Kabat numbering system,wherein the VL CDR1, VL CDR2, and VL CDR3 comprise the amino acidsequences of the VL CDR1, VL CDR2, and VL CDR3 set forth in Table 7using the Kabat numbering system, and wherein the VL FR1, VL FR2, VLFR3, and VL FR4 comprise the amino acid sequences of the VL FR1, VL FR2,VL FR3, and VL FR4 set forth in Table 9 using the Kabat numberingsystem. In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH) and avariable light chain region (VL), wherein the VH comprises a VHframework region (FR) 1, a VH CDR1, a VH FR2, a VH CDR2, a VH FR3, a VHCDR3 and a VH FR4, wherein the VL comprises a VL FR1, a VL CDR1, a VLFR2, a VL CDR2, a VL FR3, a VL CDR3, and a VL FR4, wherein the VH CDR1,VH CDR2, VH CDR3 comprise the amino acid sequences of the VH CDR1, VHCDR2, and VH CDR3 set forth in Table 1 using the Chothia numberingsystem, wherein the VH FR1, VH FR2, VH FR3 and VH FR4 comprise the aminoacid sequences of the VH FR1, VH FR2, VH FR3, and VH FR4 set forth inTable 2, 3, 4 or 5 using the Chothia numbering system, wherein the VLCDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of the VLCDR1, VL CDR2, and VL CDR3 set forth in Table 7 using the Chothianumbering system, and wherein the VL FR1, VL FR2, VL FR3, and VL FR4comprise the amino acid sequences of the VL FR1, VL FR2, VL FR3, and VLFR4 set forth in Table 9 using the Chothia numbering system. In anotherspecific embodiment, provided herein is an anti-MERTK antibody or anantigen-binding fragment thereof that specifically binds to MERTK (e.g.,human MERTK), wherein the antibody or antigen-binding fragment thereofcomprises a variable heavy chain region (VH) and a variable light chainregion (VL), wherein the VH comprises a VH framework region (FR) 1, a VHCDR1, a VH FR2, a VH CDR2, a VH FR3, a VH CDR3 and a VH FR4, wherein theVL comprises a VL FR1, a VL CDR1, a VL FR2, a VL CDR2, a VL FR3, a VLCDR3, and a VL FR4, wherein the VH CDR1, VH CDR2, VH CDR3 comprise theamino acid sequences of the VH CDR1, VH CDR2, and VH CDR3 set forth inTable 1 using the AbM numbering system, wherein the VH FR1, VH FR2, VHFR3 and VH FR4 comprise the amino acid sequences of the VH FR1, VH FR2,VH FR3, and VH FR4 set forth in Table 2, 3, 4 or 5 using the AbMnumbering system, wherein the VL CDR1, VL CDR2, and VL CDR3 comprise theamino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 set forth inTable 7 using the AbM numbering system, and wherein the VL FR1, VL FR2,VL FR3, and VL FR4 comprise the amino acid sequences of the VL FR1, VLFR2, VL FR3, and VL FR4 set forth in Table 9 using the AbM numberingsystem. In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH) and avariable light chain region (VL), wherein the VH comprises a VHframework region (FR) 1, a VH CDR1, a VH FR2, a VH CDR2, a VH FR3, a VHCDR3 and a VH FR4, wherein the VL comprises a VL FR1, a VL CDR1, a VLFR2, a VL CDR2, a VL FR3, a VL CDR3, and a VL FR4, wherein the VH CDR1,VH CDR2, VH CDR3 comprise the amino acid sequences of the VH CDR1, VHCDR2, and VH CDR3 set forth in Table 1 using the Contact numberingsystem, wherein the VH FR1, VH FR2, VH FR3 and VH FR4 comprise the aminoacid sequences of the VH FR1, VH FR2, VH FR3, and VH FR4 set forth inTable 2, 3, 4 or 5 using the Contact numbering system, wherein the VLCDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of the VLCDR1, VL CDR2, and VL CDR3 set forth in Table 7 using the Contactnumbering system, and wherein the VL FR1, VL FR2, VL FR3, and VL FR4comprise the amino acid sequences of the VL FR1, VL FR2, VL FR3, and VLFR4 set forth in Table 9 using the Contact numbering system. In anotherspecific embodiment, provided herein is an anti-MERTK antibody or anantigen-binding fragment thereof that specifically binds to MERTK (e.g.,human MERTK), wherein the antibody or antigen-binding fragment thereofcomprises a variable heavy chain region (VH) and a variable light chainregion (VL), wherein the VH comprises a VH framework region (FR) 1, a VHCDR1, a VH FR2, a VH CDR2, a VH FR3, a VH CDR3 and a VH FR4, wherein theVL comprises a VL FR1, a VL CDR1, a VL FR2, a VL CDR2, a VL FR3, a VLCDR3, and a VL FR4, wherein the VH CDR1, VH CDR2, VH CDR3 comprise theamino acid sequences of the VH CDR1, VH CDR2, and VH CDR3 set forth inTable 1 using the IMGT numbering system, wherein the VH FR1, VH FR2, VHFR3 and VH FR4 comprise the amino acid sequences of the VH FR1, VH FR2,VH FR3, and VH FR4 set forth in Table 2, 3, 4 or 5 using the IMGTnumbering system, wherein the VL CDR1, VL CDR2, and VL CDR3 comprise theamino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 set forth inTable 7 using the IMGT numbering system, and wherein the VL FR1, VL FR2,VL FR3, and VL FR4 comprise the amino acid sequences of the VL FR1, VLFR2, VL FR3, and VL FR4 set forth in Table 9 using the IMGT numberingsystem.

In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH) and avariable light chain region (VL), wherein the VH comprises a VHframework region (FR) 1, a VH CDR1, a VH FR2, a VH CDR2, a VH FR3, a VHCDR3 and a VH FR4, wherein the VL comprises a VL FR1, a VL CDR1, a VLFR2, a VL CDR2, a VL FR3, a VL CDR3, and a VL FR4, wherein the VH CDR1,VH CDR2, VH CDR3 comprise the amino acid sequences of the VH CDR1, VHCDR2, and VH CDR3 set forth in Table 1 using one numbering system,wherein the VH FR1, VH FR2, VH FR3 and VH FR4 comprise the amino acidsequences of the VH FR1, VH FR2, and VH FR4 set forth in Table 2, 3, or4 using the same numbering system, wherein the VH FR3 comprises theamino acid sequence of the VH FR3 set forth in Table 5, wherein the VLCDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of the VLCDR1, VL CDR2, and VL CDR3 set forth in Table 7 using the same numberingsystem, and wherein the VL FR1, VL FR2, VL FR3, and VL FR4 comprise theamino acid sequences of the VL FR1, VL FR2, VL FR3, and VL FR4 set forthin Table 9 using the same numbering system.

In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH) and avariable light chain region (VL), wherein the VH CDR1 comprises theamino acid sequence of SEQ ID NO: 1, the VH CDR2 comprises the aminoacid sequence of SEQ ID NO: 6, the VH CDR3 comprises the amino acidsequence of SEQ ID NO: 11, the VL CDR1 comprises the amino acid sequenceof SEQ ID NO: 68, the VL CDR2 comprises the amino acid sequence of SEQID NO: 69 and the VL CDR3 comprises the amino acid sequence of SEQ IDNO: 70.

In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH) and avariable light chain region (VL), wherein the VH CDR1 comprises theamino acid sequence of SEQ ID NO: 2, the VH CDR2 comprises the aminoacid sequence of SEQ ID NO: 7, the VH CDR3 comprises the amino acidsequence of SEQ ID NO: 12, the VL CDR1 comprises the amino acid sequenceof SEQ ID NO: 71, the VL CDR2 comprises the amino acid sequence of SEQID NO: 72 and the VL CDR3 comprises the amino acid sequence of SEQ IDNO: 73.

In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH) and avariable light chain region (VL), wherein the VH CDR1 comprises theamino acid sequence of SEQ ID NO: 3, the VH CDR2 comprises the aminoacid sequence of SEQ ID NO: 8, the VH CDR3 comprises the amino acidsequence of SEQ ID NO: 11, the VL CDR1 comprises the amino acid sequenceof SEQ ID NO: 68, the VL CDR2 comprises the amino acid sequence of SEQID NO: 69 and the VL CDR3 comprises the amino acid sequence of SEQ IDNO: 70.

In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH) and avariable light chain region (VL), wherein the VH CDR1 comprises theamino acid sequence of SEQ ID NO: 4, the VH CDR2 comprises the aminoacid sequence of SEQ ID NO: 9, the VH CDR3 comprises the amino acidsequence of SEQ ID NO: 13, the VL CDR1 comprises the amino acid sequenceof SEQ ID NO: 74, the VL CDR2 comprises the amino acid sequence of SEQID NO: 75 and the VL CDR3 comprises the amino acid sequence of SEQ IDNO: 76.

In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH) and avariable light chain region (VL), wherein the VH CDR1 comprises theamino acid sequence of SEQ ID NO: 5, the VH CDR2 comprises the aminoacid sequence of SEQ ID NO: 10, the VH CDR3 comprises the amino acidsequence of SEQ ID NO: 14, the VL CDR1 comprises the amino acid sequenceof SEQ ID NO: 77, the VL CDR2 comprises the amino acid sequence of SEQID NO: 72 and the VL CDR3 comprises the amino acid sequence of SEQ IDNO: 70.

In another specific embodiment, provided herein is an anti-MERTKantibody or an antigen-binding fragment thereof that specifically bindsto MERTK (e.g., human MERTK), wherein the antibody or antigen-bindingfragment thereof comprises a variable heavy chain region (VH) and avariable light chain region (VL), wherein the VH CDR1 comprises theamino acid sequence of SEQ ID NO: 3, the VH CDR2 comprises the aminoacid sequence of SEQ ID NO: 6, the VH CDR3 comprises the amino acidsequence of SEQ ID NO: 11, the VL CDR1 comprises the amino acid sequenceof SEQ ID NO: 68, the VL CDR2 comprises the amino acid sequence of SEQID NO: 69 and the VL CDR3 comprises the amino acid sequence of SEQ IDNO: 70.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), comprises a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 105. In anotherparticular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), comprises a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 107. In anotherparticular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), comprises a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID: 108. In another particularembodiment, a humanized anti-MERTK or an antigen-binding fragmentthereof described herein, which specifically binds to MERTK (e.g., humanMERTK), comprises a heavy chain variable region (VH) comprising theamino acid sequence of SEQ ID NO: 109.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), comprises a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID: 110.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), comprises a light chain variable region (VL)comprising the amino acid sequence of SEQ ID NO: 106. In anotherembodiment, an anti-MERTK antibody or an antigen-binding fragmentthereof, which specifically binds to MERTK (e.g., human MERTK),comprises a variable light chain region (VL) comprising the amino acidsequence of SEQ ID NO: 111.

In a specific embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),comprises a heavy chain variable region (VH) and a light chain variableregion (VL), wherein the VH comprises the amino acid sequence of SEQ IDNO: 105, and the VL comprises the amino acid sequence of SEQ ID NO: 106.In another specific embodiment, an anti-MERTK antibody orantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK) comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein the VH comprises the aminoacid sequence of SEQ ID NO: 107, and the VL comprises the amino acidsequence of SEQ ID NO: 106. In another embodiment, an anti-MERTKantibody or antigen-binding fragment thereof, which specifically bindsto MERTK (e.g., human MERTK) comprises a heavy chain variable region(VH) and a light chain variable region (VL), wherein the VH comprisesthe amino acid sequence of SEQ ID NO: 108, and the VL comprises theamino acid sequence of SEQ ID NO: 106. In another embodiment, ananti-MERTK antibody or antigen-binding fragment thereof, whichspecifically binds to MERTK (e.g., human MERTK) comprises a heavy chainvariable region (VH) and a light chain variable region (VL), wherein theVH comprises the amino acid sequence of SEQ ID NO: 110, and the VLcomprises the amino acid sequence of SEQ ID NO: 111.

In another specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK), comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein the VH comprises the aminoacid sequence of SEQ ID NO: 109, and the VL comprises the amino acidsequence of SEQ ID NO: 106 or 111.

In another specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK), comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein the VH comprises the aminoacid sequence of the VH set forth in Table 11 and the VL comprises theamino acid sequence of the VL set forth in Table 11. In another specificembodiment, a humanized anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),comprises a heavy chain variable region (VH) and a light chain variableregion (VL), wherein the VH comprises the amino acid sequence of the VHset forth in Table 12 and the VL comprises the amino acid sequence ofthe VL set forth in Table 12. In another specific embodiment, ananti-MERTK antibody or an antigen-binding fragment thereof, whichspecifically binds to MERTK (e.g., human MERTK), comprises a heavy chainvariable region (VH) and a light chain variable region (VL), wherein theVH comprises the amino acid sequence of the VH set forth in Table 13 andthe VL comprises the amino acid sequence of the VL set forth in Table13. In another specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK), comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein the VH comprises the aminoacid sequence of the VH set forth in Table 14 and the VL comprises theamino acid sequence of the VL set forth in Table 11, 12 or 13.

In certain aspects, an antibody described herein may be described by itsVH domain alone, or its VL domain alone. See, for example, Rader C etal., (1998) PNAS 95: 8910-8915, which is incorporated herein byreference in its entirety, describing the humanization of the mouseanti-αvβ3 antibody by identifying a complementing light chain or heavychain, respectively, from a human light chain or heavy chain library,resulting in humanized antibody variants having affinities as high orhigher than the affinity of the original antibody. See also, Clackson Tet al., (1991) Nature 352: 624-628, which is incorporated herein byreference in its entirety, describing methods of producing antibodiesthat bind a specific antigen by using a specific VH domain (or VLdomain) and screening a library for the complementary variable domains.See also, Kim S J & Hong H J, (2007) J Microbiol 45: 572-577, which isincorporated herein by reference in its entirety, describing methods ofproducing antibodies that bind a specific antigen by using a specific VHdomain and screening a library (e.g., human VL library) forcomplementary VL domains; the selected VL domains in turn could be usedto guide selection of additional complementary (e.g., human) VH domains.

As used herein, the terms “immunospecifically binds,”“immunospecifically recognizes,” “specifically binds,” and “specificallyrecognizes” are analogous terms in the context of antibodies and referto antibodies and antigen-binding fragments thereof that bind to anantigen (e.g., epitope or immune complex) via the antigen-binding sitesas understood by one skilled in the art, and does not excludecross-reactivity of the antibody or antigen-binding fragment with otherantigens. Any method known in the art can be used to ascertain whetherimmunospecific binding to MERTK (e.g., human MERTK) is maintained.

In specific aspects, provided herein is an anti-MERTK antibody orantigen-binding fragment thereof comprising an antibody heavy chainand/or light chain, e.g., a heavy chain alone, a light chain alone, orboth a heavy chain and a light chain. With respect to the light chain,in a specific embodiment, the light chain of an anti-MERTK antibody oran antigen-binding fragment thereof described herein is a kappa lightchain. In another specific embodiment, the light chain of an anti-MERTKantibody or an antigen-binding fragment thereof described herein is alambda light chain. In yet another specific embodiment, the light chainof an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein is a human kappa light chain or a human lambda lightchain.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises a light chain, wherein thelight chain comprises a variable light chain region (VL) and the kappalight chain constant region amino acid sequence, wherein the VLcomprises the amino acid sequence of SEQ ID NO: 106 or SEQ ID NO: 111.As used herein, the term “constant region” or “constant domain” areinterchangeable and have its meaning common in the art. The constantregion is an antibody portion, e.g., a carboxyl terminal portion of alight and/or heavy chain which is not directly involved in binding of anantibody to antigen but which can exhibit various effector functions,such as interaction with the Fc receptor. The constant region of animmunoglobulin molecule generally has a more conserved amino acidsequence relative to an immunoglobulin variable domain.

In a specific embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), comprises a light chain, wherein the light chaincomprises a variable light chain region (VL) and a human kappa or lambdalight chain constant region, wherein the VL comprises the amino acidsequence of SEQ ID NO: 106 or SEQ ID NO: 111. Non-limiting examples ofhuman constant region sequences have been described in the art, e.g.,see Kabat E A et al., (1991).

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises a light chain, wherein thelight chain comprises the amino acid sequence of SEQ ID NO: 113 or SEQID NO: 118.

With respect to the heavy chain, in a specific embodiment, the heavychain of an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein can be a human alpha (α), delta (δ), epsilon (ε), gamma(γ) or mu (μ) heavy chain. In a specific embodiment, an anti-MERTKantibody or an antigen-binding fragment thereof, which specificallybinds to MERTK (e.g., human MERTK), comprises a heavy chain, wherein theheavy chain comprises the constant region or a portion thereof (e.g.C_(H)1, C_(H)2 or C_(H)3 or a combination thereof) described herein orknown in the art, and a variable heavy chain region (VH) of ananti-MERTK antibody described herein (e.g., SEQ ID NO: 105, SEQ ID NO:107, SEQ ID NO: 108, 109 or SEQ ID NO: 110).

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),comprises a heavy chain, wherein the heavy chain comprises a variableheavy chain region (VH) of an anti-MERTK antibody described herein(e.g., SEQ ID NO: 105, 107, 108, 109 or 110), and the constant region orportion thereof (e.g. C_(H)1, C_(H)2 or C_(H)3 or a combination thereof)of the human gamma heavy chain constant region variable region. In aspecific embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK)comprises the amino acid sequence of SEQ ID NO: 112, SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117. Non-limiting examples ofhuman constant region sequences have been described in the art, e.g.,see Kabat E A et al., (1991) supra.

In a specific embodiment, an anti-MERTK antibody described herein, whichspecifically binds to MERTK (e.g., human MERTK), comprises (i) a heavychain comprising the amino acid sequence of SEQ ID NO: 112 and (ii) alight chain comprising the amino acid of SEQ ID NO: 113. In a specificembodiment, an anti-MERTK antibody described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises (i) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 114 and (ii) a lightchain comprising the amino acid of SEQ ID NO: 113. In a specificembodiment, an anti-MERTK antibody described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises (i) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 115 and (ii) a lightchain comprising the amino acid sequence of SEQ ID NO: 113. In aspecific embodiment, an anti-MERTK antibody described herein, whichspecifically binds to MERTK (e.g., human MERTK), comprises (i) a heavychain comprising the amino acid sequence of SEQ ID NO: 116 and (ii) alight chain comprising the amino acid of SEQ ID NO: 113. In a specificembodiment, an anti-MERTK antibody described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises (i) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 117 and (ii) a lightchain comprising the amino acid of SEQ ID NO: 118.

In a specific embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof described herein, which immunospecifically binds toMERTK (e.g., human MERTK) comprises a heavy chain variable region (VH)and a light chain variable region (VL) comprising any amino acidsequences described herein, and wherein the constant regions comprisethe amino acid sequences of the constant regions of a human IgG, IgE,IgM, IgD, IgA or IgY immunoglobulin molecule. In another specificembodiment, an anti-MERTK antibody or antigen-binding fragment thereofdescribed herein, which immunospecifically binds to MERTK (e.g., humanMERTK) comprises a VH and a VL comprising any amino acid sequencesdescribed herein, and wherein the constant regions comprise the aminoacid sequences of the constant regions (e.g., those set forth in Tables11, 12, 13 or 15) of a human IgG, IgE, IgM, IgD, IgA or IgYimmunoglobulin molecule, any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂), or any subclass (e.g., IgG_(2a) and IgG_(2b)) ofimmunoglobulin molecule.

In certain embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an anti-MERTKantibody or an antigen-binding fragment thereof described herein toalter one or more functional properties of the antibody.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an anti-MERTKantibody or an antigen-binding fragment thereof described herein (e.g.,C_(H)2 domain (residues 231-340 of human IgG₁) and/or C_(H)3 domain(residues 341-447 of human IgG₁) and/or the hinge region, with numberingaccording to the Kabat numbering system (e.g., the EU index in Kabat))to increase or decrease the affinity of the antibody or anantigen-binding fragment thereof for an Fc receptor (e.g., an activatedFc receptor) on the surface of an effector cell. Mutations in the Fcregion of an anti-MERTK antibody or antigen-binding fragment thereofthat decrease or increase the affinity of an antibody for an Fcreceptor, and techniques for introducing such mutations into the Fcreceptor or fragment thereof are known to one of skill in the art.Examples of mutations in the Fc receptor of an anti-MERTK antibody orantigen-binding fragment thereof that can be made to alter the affinityof the antibody or an antigen-binding fragment thereof for an Fcreceptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporatedherein by reference.

In some embodiments, the antibody or antigen-binding fragment thereofdescribed herein comprises a glycosylated constant region. In someembodiments, the antibody or antigen-binding fragment thereof describedherein comprises a non-glycosylated constant region. Antibodies withreduced fucose content have been reported to have an increased affinityfor Fc receptors, such as, e.g., FcγRIIIa. Accordingly, in certainembodiments, the antibodies or antigen-binding fragments thereofdescribed herein have reduced fucose content or no fucose content. Suchantibodies or antigen-binding fragments thereof can be produced usingtechniques known to one skilled in the art. For example, the antibodiesor antigen-binding fragments thereof can be expressed in cells deficientor lacking the ability of fucosylation. In a specific example, celllines with a knockout of both alleles of α1,6-fucosyltransferase can beused to produce antibodies with reduced fucose content. The Potelligent®system (Lonza) is an example of such a system that can be used toproduce antibodies or antigen-binding fragments thereof with reducedfucose content. Alternatively, antibodies or antigen-binding fragmentswith reduced fucose content or no fucose content can be produced by,e.g.: (i) culturing cells under conditions which prevent or reducefucosylation; (ii) posttranslational removal of fucose (e.g., with afucosidase enzyme); (iii) post-translational addition of the desiredcarbohydrate, e.g., after recombinant expression of a non-glycosylatedglycoprotein; or (iv) purification of the glycoprotein so as to selectfor antibodies or antigen-binding fragments thereof which are notfucsoylated. See, e.g., Longmore G D & Schachter H (1982) Carbohydr Res100: 365-92 and Imai-Nishiya H et al., (2007) BMC Biotechnol. 7: 84 formethods for producing antibodies or antigen-binding fragments thereofwith no fucose content or reduced fucose content.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises a heavy chain or a lightchain, wherein (i) the heavy chain comprises (a) a variable regioncomprising the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: 107,SEQ ID NO: 108 or SEQ ID NO: 109 and (b) a constant heavy chain domaincomprising the amino acid sequence of the constant domain of a humanIgG; or (ii) the light chain comprises (a) a variable region comprisingthe amino acid sequence of SEQ ID NO: 106 and (b) a constant light chaindomain comprising the amino acid sequence of the constant domain of ahuman light chain.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises a heavy chain and a lightchain, wherein (i) the heavy chain comprises (a) a variable regioncomprising the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: 107,SEQ ID NO: 108, or SEQ ID NO: 109 and (b) a constant heavy chain domaincomprising the amino acid sequence of the constant domain of a humanIgG; and (ii) the light chain comprises a variable region comprising theamino acid sequence of SEQ ID NO: 106, and (b) a constant light chaindomain comprising the amino acid sequence of the constant domain of ahuman kappa light chain.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, which specificallybinds to MERTK (e.g., human MERTK), comprises a heavy chain or a lightchain, wherein (i) the heavy chain comprises (a) a variable regioncomprising the amino acid sequence of SEQ ID NO: 110 and (b) a constantheavy chain domain comprising the amino acid sequence of the constantdomain of a human IgG; or (ii) the heavy chain comprises (a) a variableregion comprising the amino acid sequence of SEQ ID NO: 111 and (b) aconstant light chain domain comprising the amino acid sequence of theconstant domain of a human kappa light chain. In another particularembodiment, an anti-MERTK antibody or an antigen-binding fragmentthereof described herein, which specifically binds to MERTK (e.g., humanMERTK), comprises a heavy chain and a light chain, wherein (i) the heavychain comprises (a) a variable region comprising the amino acid sequenceof SEQ ID NO: 110 and (b) a constant heavy chain domain comprising theamino acid sequence of the constant domain of a human IgG; and (ii) thelight chain comprises (a) a variable region comprising the amino acidsequence of SEQ ID NO: 111 and (b) a constant light chain domaincomprising the amino acid sequence of the constant domain of a humankappa light chain.

The determination of percent identity between two sequences (e.g., aminoacid sequences or nucleic acid sequences) can also be accomplished usinga mathematical algorithm. A specific, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm ofKarlin S & Altschul S F (1990) PNAS 87: 2264-2268,modified as in Karlin S & Altschul S F (1993) PNAS 90: 5873-5877. Suchan algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul S F et al., (1990) J Mol Biol 215: 403. BLAST nucleotidesearches can be performed with the NBLAST nucleotide program parametersset, e.g., for score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules described herein. BLAST proteinsearches can be performed with the XBLAST program parameters set, e.g.,to score 50, wordlength=3 to obtain amino acid sequences homologous to aprotein molecule described herein. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul S F et al., (1997) Nuc Acids Res 25: 3389 3402. Alternatively,PSI BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g.,National Center for Biotechnology Information (NCBI) on the worldwideweb, ncbi.nlm.nih.gov). Another specific, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

In certain embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),comprises a VH having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the amino acid sequence of the VH of SEQ ID NO: 105, SEQ IDNO: 107, or SEQ ID NO: 108 or SEQ ID NO: 109. In some embodiments, ananti-MERTK antibody or an antigen-binding fragment thereof comprises aVH having at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of the VH of SEQ ID NO:110.

In certain embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof, which immunospecifically binds to MERTK (e.g., humanMERTK), comprises a VL having at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of the VL of SEQ ID NO:106. In certain embodiments, an anti-MERTK antibody or anantigen-binding fragment thereof comprises a VL having at least 99%sequence identity to the VH of SEQ ID NO: 111.

In certain embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),comprises: (i) a VH domain having at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to the amino acid sequence of the VH domain of SEQID NO: 105, SEQ ID NO: 107, or SEQ ID NO: 108 or SEQ ID NO: 109; and(ii) a VL domain having at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% sequence identity to the amino acid sequenceof the VL domain of SEQ ID NO: 106.

In certain embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), comprises (i) a VH domain having at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to the VHdomain of SEQ ID NO: 110, and (ii) a VL domain having at least 99%sequence identity to the VL domain of SEQ ID NO: 111.

In another aspect, provided herein are antibodies that bind the same oran overlapping epitope of MERTK (e.g., human MERTK) as an antibodydescribed herein. As used herein, an “epitope” is a term in the art andrefers to a localized region of an antigen to which an antibody canspecifically bind. An epitope can be, for example, contiguous aminoacids of a polypeptide (linear or contiguous epitope) or an epitope can,for example, come together from two or more non-contiguous regions of apolypeptide or polypeptides (conformational, non-linear, discontinuous,or non-contiguous epitope). In certain embodiments, the epitope of anantibody can be determined by, e.g., NMR spectroscopy, X-ray diffractioncrystallography studies, ELISA assays, hydrogen/deuterium exchangecoupled with mass spectrometry (e.g., MALDI mass spectrometry),array-based oligo-peptide scanning assays, and/or mutagenesis mapping(e.g., site-directed mutagenesis mapping). For X-ray crystallography,crystallization may be accomplished using any of the known methods inthe art (e.g., Giege R et al., (1994) Acta Crystallogr D BiolCrystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) JBiol Chem 251: 6300-6303). Antibody:antigen crystals may be studiedusing well known X-ray diffraction techniques and may be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc.; see e.g. Meth Enzymol (1985) volumes 114 &115, eds Wyckoff H W et al.,; U.S. Patent Application No. 2004/0014194),and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter C W;Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies may be accomplished using anymethod known to one of skill in the art. See, e.g., Champe M et al.,(1995) and Cunningham B C & Wells J A (1989) for a description ofmutagenesis techniques, including alanine scanning mutagenesistechniques. In addition, antibodies that recognize and bind to the sameor overlapping epitopes of MERTK (e.g., human MERTK) can be identifiedusing routine techniques such as an immunoassay, for example, by showingthe ability of one antibody to block the binding of another antibody toa target antigen, i.e., a competitive binding assay. Competition bindingassays also can be used to determine whether two antibodies have similarbinding specificity for an epitope. Competitive binding can bedetermined in an assay in which the immunoglobulin under test inhibitsspecific binding of a reference antibody to a common antigen, such asMERTK. Numerous types of competitive binding assays are known, forexample: solid phase direct or indirect radioimmunoassay (RIA), solidphase direct or indirect enzyme immunoassay (EIA), sandwich competitionassay (see Stahli C et al., (1983) Methods Enzymol 9: 242-253); solidphase direct biotin-avidin EIA (see Kirkland T N et al., (1986) JImmunol 137: 3614-9); solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see Harlow E & Lane D, (1988) Antibodies:A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labelRIA using 1-125 label (see Morel G A et al., (1988) Mol Immunol 25(1):7-15); solid phase direct biotin-avidin EIA (Cheung R C et al., (1990)Virology 176: 546-52); and direct labeled RIA. (Moldenhauer G et al.,(1990) Scand J Immunol 32: 77-82). Typically, such an assay involves theuse of purified antigen (e.g., MERTK (e.g., human MERTK)) bound to asolid surface or cells bearing either of these, an unlabeled testimmunoglobulin and a labeled reference immunoglobulin. Competitiveinhibition can be measured by determining the amount of label bound tothe solid surface or cells in the presence of the test immunoglobulin.Usually the test immunoglobulin is present in excess. Usually, when acompeting antibody is present in excess, it will inhibit specificbinding of a reference antibody to a common antigen by at least 50-55%,55-60%, 60-65%, 65-70% 70-75% or more. A competition binding assay canbe configured in a large number of different formats using eitherlabeled antigen or labeled antibody. In a common version of this assay,the antigen is immobilized on a 96-well plate. The ability of unlabeledantibodies to block the binding of labeled antibodies to the antigen isthen measured using radioactive or enzyme labels. For further detailssee, for example, Wagener C et al., (1983) J Immunol 130: 2308-2315;Wagener C et al., (1984) J Immunol Methods 68: 269-274; Kuroki M et al.,(1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest21: 523-538; Kuroki M et al., (1992) Hybridoma 11: 391-407 andAntibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra, pp.386-389.

In certain aspects, competition binding assays can be used to determinewhether an antibody is competitively blocked, e.g., in a dose dependentmanner, by another antibody for example, an antibody binds essentiallythe same epitope, or overlapping epitopes, as a reference antibody, whenthe two antibodies recognize identical or sterically overlappingepitopes in competition binding assays such as competition ELISA assays,which can be configured in a number of different formats, using eitherlabeled antigen or labeled antibody. In a particular embodiment, anantibody can be tested in competition binding assays with an antibodydescribed herein (e.g., antibody z10, z11, or z13).

In another aspect, provided herein are antibodies that compete (e.g., ina dose dependent manner) for binding to MERTK (e.g., human MERTK) withan anti-MERTK antibody described herein (e.g., z10, z11, z13 or xAb) asdetermined using assays known to one of skill in the art or describedherein (e.g., ELISA competitive assays or surface plasmon resonance). Inanother aspect, provided herein are antibodies that competitivelyinhibit (e.g., in a dose dependent manner) an anti-MERTK antibodydescribed herein (e.g., z10, z11, z13 or xAb) from binding to MERTK(e.g., human MERTK), as determined using assays known to one of skill inthe art or described herein (e.g., ELISA competitive assays, orsuspension array or surface plasmon resonance).

In certain embodiments, provided herein is an anti-MERTK antibody thatcompetes with an antibody described herein for binding to MERTK (e.g.,human MERTK) to the same extent that the antibody described hereinself-competes for binding to MERTK (e.g., human MERTK). In certainembodiments, provided herein is a first antibody that competes with ananti-MERTK antibody described herein for binding to MERTK (e.g., humanMERTK), wherein the competition is exhibited as reduced binding of thefirst antibody to MERTK (e.g., human MERTK) by more than 80% (e.g., 85%,90%, 95%, or 98%, or between 80% to 85%, 80% to 90%, 85% to 90%, or 85%to 95%).

In specific aspects, provided herein is an anti-MERTK antibody whichcompetes (e.g., in a dose dependent manner) for specific binding toMERTK (e.g., human MERTK), with an antibody comprising a VH domaincomprising the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: 107,SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110, and a VL domaincomprising the amino acid sequence of SEQ ID NO: 106 or SEQ ID NO: 111

In a specific embodiment, an anti-MERTK antibody (e.g., a humanizedantibody or a human antibody) or an antigen-binding fragment thereofthat specifically binds to the same or an overlapping epitope of anantibody comprising a VH domain having an amino acid sequence of SEQ IDNO: 105 and a VL domain having an amino acid sequence of SEQ ID NO: 106.In another specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein immunospecificallybinds to the same or an overlapping epitope of an antibody comprising aVH domain having an amino acid sequence of SEQ ID NO: 107, and a VLdomain having an amino acid sequence of SEQ ID NO: 106. In anotherspecific embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein immunospecifically binds to the sameor an overlapping epitope of an antibody comprising a VH domain havingan amino acid sequence of SEQ ID NO: 108, and a VL domain having anamino acid sequence of SEQ ID NO: 106. In another specific embodiment,an-MERTK antibody or an antigen-binding fragment thereof describedherein immunospecifically binds to the same or an overlapping epitope ofan antibody comprising a VH domain having an amino acid sequence of SEQID NO: 110, and a VL domain having an amino acid sequence of SEQ ID NO:111. Assays known to one of skill in the art or described herein (e.g.,X-ray crystallography, ELISA assays, etc.) can be used to determine iftwo antibodies bind to the same epitope. In specific embodiments, anantibody or antigen-binding fragment thereof that competes with anantibody described herein for binding to MERTK (e.g., human MERTK) isnot a murine antibody. In specific embodiments, an antibody orantigen-binding fragment thereof that binds to the same or anoverlapping epitope of an antibody described herein (e.g., human MERTK)is not a murine antibody.

Affinity can be measured and/or expressed in a number of ways known inthe art, including, but not limited to, equilibrium dissociationconstant (K_(D)), and equilibrium association constant (KA). The K_(D)can be determined by techniques known to one of ordinary skill in theart, such as biolayer interferometry or surface plasmon resonance.

In certain embodiments, an anti-MERTK antibody (e.g. a humanizedantibody) or an antigen-binding fragment thereof described herein, thatcompetes with an antibody described herein for binding to MERTK (e.g.,human MERTK), or an anti-MERTK antibody or antigen-binding fragmentthereof that binds to the same or an overlapping epitope of ananti-MERTK antibody described herein, binds to MERTK (e.g. human MERTK)with a K_(D) of about 8 nM, 7 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.75 nM, 0.5 nM, 0.25 nM, or 0.1 nM. Insome embodiments, an anti-MERTK antibody or an antigen-binding fragmentthereof described herein, that competes with an antibody describedherein for binding to MERTK (e.g., human MERTK), or an anti-MERTKantibody or antigen-binding fragment thereof that binds to the same oran overlapping epitope of an antibody described herein, binds to MERTK(e.g., human MERTK) with a K_(D) of 0.1 nM to 10 nM, 0.5 nM to 10 nM, 1nM to 10 nM, 2 nM to 10 nM, 2 nM to 5 nM, 4 nM to 10 nM in an assay suchas described herein or known to one of skill in the art. In someembodiments, an anti-MERTK antibody or an antigen-binding fragmentthereof described herein that competes with an antibody described hereinfor binding to MERTK (e.g., human MERTK), or an anti-MERTK antibody orantigen-binding fragment thereof that binds to the same or anoverlapping epitope of an antibody described herein, binds to MERTK(e.g., human MERTK) with a K_(D) avidity of about 2 pM to 8 pM using anassay described herein or known to one of skill in the art.

In a specific embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein binds to MERTK (e.g., human MERTK)with a K_(D) of about 3 nM in an assay described herein or known to oneof skill in the art. In some embodiments, an anti-MERTK antibody or anantigen-binding fragment thereof described herein binds to MERTK (e.g.,human MERTK) with a K_(D) avidity of about 1 pM to 15 pM in an assaydescribed herein or known in the art. In another specific embodiment, ananti-MERTK antibody or an antigen-binding fragment thereof describedherein binds to MERTK (e.g., human MERTK) with a K_(D) avidity of about1.46 nM in an assay described herein or known to one of skill in theart. In another specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein binds to MERTK (e.g.,human MERTK) with a K_(D) avidity of about 5.74 pM in an assay describedherein or known to one of skill in the art. In another specificembodiment, an anti-MERTK antibody or an antigen-binding fragmentthereof described herein binds to MERTK (e.g., human MERTK) with a K_(D)avidity of about 3.68 pM in an assay described herein or known to one ofskill in the art. In another specific embodiment, an anti-MERTK antibodyor an antigen-binding fragment thereof described herein binds to MERTK(e.g., human MERTK) with a K_(D) avidity of about 2.99 pM in assaydescribed herein or known to one of skill in the art. As used herein,the terms “about” when used to modify a numeric value or numeric range,indicate that deviations of 5% to 10% above and 5% to 10% below thevalue or range remain within the intended meaning of the recited valueor range.

In a specific embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof binds to MERTK (e.g. human MERTK) with an EC₅₀ of 1 nMto 20 nM as assessed by an assay described herein or known in the art.In another specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof binds to MERTK (e.g., human MERTK) withan EC₅₀ of 1 nM to 10 nM as assessed by an assay described herein orknown in the art. In anther specific embodiment, an anti-MERTK antibodyor antigen-binding fragment thereof binds to MERTK (e.g., human MERTK)with an EC₅₀ of about 1 nM, about 2 nM, about 3 nM, about 4 NM, about 5nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM or about 10 nM asassessed by an assay described herein or known in the art.

In a specific embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof is one described in section 6, infra.

5.1.2. Functional Characteristics

In some aspects, an anti-MERTK antibody or an antigen-binding fragmentthereof described herein, which specifically binds to MERTK (e.g., humanMERTK), decreases expression levels of human MERTK on cancer cells (e.g.SKMEL5 melanoma cells) by more than 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% as assessed by an assayknown to one of skill in the art or described herein. In some aspects,an anti-MERTK antibody or an antigen-binding fragment thereof describedherein, which specifically binds to human MERTK, decreases expressionlevels of human MERTK on human M2 macrophages by more than 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%as assessed by an assay known to one of skill in the art or describedherein. In specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof binds to MERTK (e.g., human MERTK, butnot human Axl, human Tyro3, or murine MERTK, as assessed by methodsdescribed herein or known in the art.

In some embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof binds to human MERTK and cynomolgus monkey MERTK.

In some aspects, an anti-MERTK antibody or an antigen-binding fragmentthereof described herein, which specifically binds to MERTK (e.g., humanMERTK), induces cytokine secretion responses by M2 macrophages, CD14+monocytes, or both such as set forth in FIG. 10. In certain aspects, ananti-MERTK antibody or an antigen-binding fragment thereof alters theexpression of certain cytokines (e.g. the antibody or antigen-bindingfragment thereof increases expression of certain cytokines), such as setforth in FIG. 10, by M2 macrophages, CD14+ monocytes, or both by morethan 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80% or 85% as assessed by an assay known to one of skill in the artor described herein.

In some embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),blocks (e.g., partially or completely) the level of MERTKphosphorylation induced by Gas-6 in human MERTK expressing cells, suchas, e.g., cancer cells (e.g., a melanoma cells), M2 macrophages or both,in an assay known to one of skill in the art. In specific embodiments,an anti-MERTK antibody or an antigen-binding fragment thereof, whichspecifically binds to MerTK (e.g., human MERTK), reduces the level ofhuman MERTK phosphorylation induced by Gas-6 in human MERTK expressingcells, such as, e.g., cancer cells (e.g., a melanoma cells), M2macrophages or both, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% in an assay known to one ofskill in the art.

In certain embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),induces degradation of human MERTK on human MERTK expressing cells, suchas, e.g., cancer cells (e.g., a melanoma cells), M2 macrophages or both.In specific embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),reduces the level of human MERTK on the surface of human MERTKexpressing cells, such as, e.g., cancer cells (e.g., a melanoma cells),M2 macrophages or both, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% in an assay describedherein or known to one of skill in the art.

In certain embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),induces internalization of human MERTK on human MERTK expressing cells,such as, e.g., cancer cells (e.g., a melanoma cells), M2 macrophages orboth. In specific embodiments, an anti-MERTK antibody or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK), increases the internalization of human MERTK byhuman MERTK expressing cells, such as, e.g., cancer cells (e.g., amelanoma cells), M2 macrophages or both, by at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% in an assaydescribed herein or known to one of skill in the art.

In some embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),inhibits (e.g., partially or completely) colony formation by cancercells (e.g., a melanoma cells) in an assay described herein or known toone of skill in the art. In specific embodiments, an anti-MERTK antibodyor an antigen-binding fragment thereof, which specifically binds toMERTK (e.g., human MERTK), reduces the level of colony formation bycancer cells (e.g., a melanoma cells) by at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% in an assaydescribed herein or known to one of skill in the art. In some aspects,an anti-MERTK antibody or an antigen-binding fragment thereof describedherein, which immunospecifically binds to MERTK (e.g., human MERTK),reduces the colony forming ability of cancer cells (e.g., SKMEL5melanoma cells) by more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% as assessed by an assay knownto one of skill in the art or described herein.

In some aspects, an anti-MERTK antibody or an antigen-binding fragmentthereof described herein, which specifically binds to MERTK (e.g., humanMERTK), prevents Gas6-induced AKT phosphorylation in an assay describedherein or known in the art. In certain embodiments, an anti-MERTKantibody or antigen-binding fragment thereof described herein, whichspecifically binds to MERTK (e.g., human MERTK), competes with Gas-6(e.g., human Gas-6) for binding to human MERTK. In some embodiments, ananti-MERTK antibody or antigen-binding fragment thereof described hereininhibits (e.g., completely inhibits or only partially inhibits) Gas-6from binding to human MERTK. In some embodiments, an anti-MERTK antibodyor an antigen-binding fragment thereof described herein, whichspecifically binds to MERTK (e.g., human MERTK), inhibits the binding ofGas-6 (e.g., human or mouse Gas-6) to human MERTK by more than 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%as assessed by an assay known to one of skill in the art or describedherein. In a specific embodiment, the assay that is used to assessinhibition of binding of Gas-6 (e.g., human Gas-6) to human MERTK in thepresence of an anti-MERTK antibody or antigen-binding fragment thereofdescribed herein, is antibody the capture ELISA known to one of skill inthe art, such as described in International Publication No.WO2016/106221 (e.g., Example 1 of International Publication No.WO2016/106221).

In some embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g, human MERTK),reduces the level of AKT phosphorylation induced by Gas-6 in human MERTKexpressing cancer cells (e.g., a melanoma cells) by at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%in an assay described herein or known to one of skill in the art.

In specific embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), inhibits angiogenesis within tumors. In someembodiments, the inhibition of angiogenesis is by at least 10%, 15% 20%,25%, 30%, 35%, 40%, 45%, 50%, 55, 60%, 65%, 70%, 75%, 80% or 85%. In aspecific embodiment, the inhibition of angiogenesis is at least 50%,55%, 60%, 65%, or 70%. Inhibition of angiogenesis can be assessed bymethods described herein and/or known to one of skill in the art. Theinhibition can be relative to the level of angiogenesis without anyantibody or with an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to MERTK).

In certain embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK), inhibits tumor progression. The inhibition of tumorprogression by at least 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55,60%, 65%, 70%, 75%, 80% or 85%. Tumor progression can be assessed bymethods known to one of skill in the art. The tumor progression can berelative to the cancer status without any antibody or with an unrelatedantibody (e.g., an antibody that does not specifically bind to humanMERTK). In a specific embodiment, an anti-MERTK antibody or anantigen-binding region thereof has one, two, three or more of thecharacteristics of an antibody described in Section 6, infra.

5.1.3. Bispecific Antibodies

In a specific aspect, provided herein is a bispecific antibodycomprising two different antigen binding regions, wherein one of thebinding regions specifically binds to human MERTK and the other bindingregion binds to another antigen of interest, and wherein the bindingregion that specifically binds to MERTK (e.g., human MERTK) is ananti-MERTK antibody or an antigen-binding fragment thereof describedherein. In a specific embodiment, provided herein is a bispecificantibody comprising two different antigen binding regions, wherein oneof the binding regions specifically binds to MERTK (e.g., human MERTK)and the other binding region binds to another antigen of interest, andwherein the binding region that specifically binds to MERTK (e.g., humanMERTK) comprises the variable heavy chain region of an anti-MERTKantibody or an antigen-binding fragment thereof described herein. Inanother specific embodiment, provided herein is a bispecific antibodycomprising two different antigen binding regions, wherein one of thebinding regions specifically binds to MERTK (e.g., human MERTK) and theother binding region binds to another antigen of interest, and whereinthe binding region that specifically binds to MERTK (e.g., human MERTK)comprises the variable heavy chain region amino acid sequence ofantibody z10 set forth in Table 11. In another specific embodiment,provided herein is a bispecific antibody comprising two differentbinding regions, wherein one of the binding regions specifically bindsto MERTK (e.g., human MERTK) and the other binding region binds toanother antigen of interest, and wherein the binding region thatspecifically binds to MERTK (e.g., human MERTK) comprises the variableheavy chain region amino acid sequence of antibody z11 set forth inTable 12. In another specific embodiment, provided herein is abispecific antibody comprising two different antigen binding regions,wherein one of the binding regions specifically binds to MERTK (e.g.,human MERTK) and the other binding region binds to another antigen ofinterest, and wherein the binding region that specifically binds toMERTK (e.g., human MERTK) comprises the variable heavy chain regionamino acid sequence of antibody z13 set forth in Table 13. In anotherspecific embodiment, provided herein is a bispecific antibody comprisingtwo different binding regions, wherein one of the binding regionsspecifically binds to MERTK (e.g., human MERTK) and the other bindingregion binds to another antigen of interest, and wherein the bindingregion that specifically binds to MERTK (e.g., human MERTK) comprises avariable heavy chain region comprising the amino acid sequence set forthin Table 14.

In another specific embodiment, provided herein is a bispecific antibodycomprising two different antigen binding regions, wherein one of thebinding regions specifically binds to MERTK (e.g., human MERTK) and theother binding region binds to another antigen of interest, and whereinthe binding region that specifically binds to MERTK (e.g., human MERTK)comprises the variable light chain region of an anti-MERTK antibody oran antigen-binding fragment thereof described herein. In anotherspecific embodiment, provided herein is a bispecific antibody comprisingtwo different antigen binding regions, wherein one of the bindingregions specifically binds to MERTK (e.g., human MERTK) and the otherbinding region binds to another antigen of interest, and wherein thebinding region that specifically binds to MERTK (e.g., human MERTK)comprises the variable light chain region amino acid sequence ofantibody z10 set forth in Table 11. In another specific embodiment,provided herein is a bispecific antibody comprising two differentantigen binding regions, wherein one of the binding regions specificallybinds to MERTK (e.g., human MERTK) and the other binding region binds toanother antigen of interest, and wherein the binding region thatspecifically binds to MERTK (e.g., human MERTK) comprises the variablelight chain region amino acid sequence of antibody z11 set forth inTable 12. In another specific embodiment, provided herein is abispecific antibody comprising two different antigen binding regions,wherein one of the binding regions specifically binds to MERTK (e.g.,human MERTK) and the other binding region binds to another antigen ofinterest, and wherein the binding region that specifically binds toMERTK (e.g., human MERTK) comprises the variable light chain regionamino acid sequence of antibody z13 set forth in Table 13. In anotherspecific embodiment, provided herein is a bispecific antibody comprisingtwo different binding regions, wherein one of the binding regionsspecifically binds to MERTK (e.g., human MERTK) and the other bindingregion binds to another antigen of interest, and wherein the bindingregion that specifically binds to MERTK (e.g., human MERTK) comprises avariable light chain region, wherein the variable light chain regioncomprises the amino acid sequence of the VL set forth in Table 11, 12 or13.

In another specific embodiment, provided herein is a bispecific antibodycomprising two different antigen binding regions, wherein one of thebinding regions specifically binds to MERTK (e.g., human MERTK) and theother binding region binds to another antigen of interest, and whereinthe binding region that specifically binds to MERTK (e.g., human MERTK)comprises the variable light chain region and the variable heavy chainregion of an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein. In another specific embodiment, provided herein is abispecific antibody comprising two different antigen binding regions,wherein one of the binding regions specifically binds to MERTK (e.g.,human MERTK) and the other binding region binds to another antigen ofinterest, and wherein the binding region that specifically binds toMERTK (e.g., human MERTK) comprises the variable light chain region andthe variable heavy chain region amino acid sequences of antibody z10 setforth in Table 11. In another specific embodiment, provided herein is abispecific antibody comprising two different antigen binding regions,wherein one of the binding regions specifically binds to MERTK (e.g.,human MERTK) and the other binding region binds to another antigen ofinterest, and wherein the binding region that specifically binds toMERTK (e.g., human MERTK) comprises the variable light chain region andthe variable heavy chain region amino acid sequences of antibody z11 setforth in Table 12. In another specific embodiment, provided herein is abispecific antibody comprising two different antigen binding regions,wherein one of the binding regions specifically binds to MERTK (e.g.,human MERTK) and the other binding region binds to another antigen ofinterest, and wherein the binding region that specifically binds toMERTK (e.g., human MERTK) comprises the variable light chain region andthe variable heavy chain region amino acid sequences of antibody z13 setforth in Table 13. In another specific embodiment, provided herein is abispecific antibody comprising two different antigen binding regions,wherein one of the binding regions specifically binds to MERTK (e.g.,human MERTK) and the other binding region binds to another antigen ofinterest, and wherein the binding region that specifically binds toMERTK (e.g., human MERTK) comprises a variable light chain region and avariable heavy chain region, wherein the variable heavy chain regioncomprises the amino acid sequence set forth in Table 14 and the variablelight chain region comprises the VL amino acid sequence set forth inTable 11, 12 or 13.

In certain embodiments, the antigen of interest to which the otherbinding region of a bispecific antibody described herein binds isantigen present on an immune cell (e.g., a T cell, an NK cell, ordendritic cell). In some embodiments, the antigen of interest to whichthe other binding region of a bispecific antibody described herein bindsis an immune checkpoint receptor (e.g., PD-1, PD-L1, CTLA-4, LAG3, Tim3,ICOS, CD40, GITR, or OX40). In a specific embodiment, the other bindingregion of a bispecific antibody described herein comprises an agonisticanti-GITR antibody, an agonistic anti-OX40 antibody or an agonisticanti-CD40 antibody. In specific embodiments, the other binding region ofa bispecific antibody comprises a blocking antibody or anantigen-binding fragment thereof that binds to PD-1, PD-L1, CTLA-4 orLAG3. In certain embodiments, the antigen of interest to which the otherbinding region of a bispecific antibody described herein binds is a Tcell receptor. In a specific embodiment, the antigen of interest towhich the other binding region of a bispecific antibody described hereinbinds is CD3, PD-L1, LRP1, LRP8, TGF-β, NKGD2 or TIGIT. In someembodiments, the antigen of interest is a tumor-associated antigen.

In a specific embodiment, the other binding region of a bispecificantibody described herein is nivolumab or an antigen-binding fragmentthereof. In another specific embodiment, the other binding region of abispecific antibody described herein is lambrolizumab or anantigen-binding fragment thereof. In another specific embodiment, theother binding region of a bispecific antibody described herein isMEDI-4736 or an antigen-binding fragment thereof. In another specificembodiment, the other binding region of a bispecific antibody describedherein is ipilimumab or an antigen-binding fragment thereof. In anotherspecific embodiment, the other binding region of a bispecific antibodydescribed herein is MPDL-3280A or an antigen-binding fragment thereof.In another specific embodiment, the other binding region of a bispecificantibody described herein is pidilizumab or an antigen-binding fragmentthereof. In another specific embodiment, the other binding region of abispecific antibody described herein is avelumab or an antigen-bindingfragment thereof. In another specific embodiment, the other bindingregion of a bispecific antibody described herein is pembrolizumab or anantigen-binding fragment thereof.

5.2. Antibody-Drug Conjugates.

Provided herein are antibody-drug conjugates comprising: (a) an antibodymoiety that is an anti-MERTK antibody or an antigen-binding fragmentthereof described herein, which specifically binds to MERTK (e.g., humanMERTK); (b) one or more drug moieties, each drug moiety being acytotoxic agent; and (c) optionally a linker; wherein the cytotoxicagent is conjugated directly to the antibody moiety or is conjugated tothe antibody moiety via the linker. The term “conjugated” as used inthis disclosure shall mean covalently bound, which can be directly orvia an intervening covalently bound structure. The antibody moiety of anantibody-drug conjugate provided herein may be an antibody described insection 5.1 or section 6. An antibody-drug conjugate may be anantibody-drug conjugate described in section 6.

In certain embodiments, the antibody-drug conjugate provided herein hasa molar ratio of the antibody moiety to the drug moiety that is between1:1 and 1:20. In specific embodiments, the antibody-drug conjugateprovided herein has a molar ratio of the antibody moiety to the drugmoiety that is between 1:1 and 1:15. In specific embodiments, theantibody-drug conjugate provided herein has a molar ratio of theantibody moiety to the drug moiety that is between 1:1 and 1:12. Inspecific embodiments, the antibody-drug conjugate provided herein has amolar ratio of the antibody moiety to the drug moiety that is between1:1 and 1:8. In preferred embodiments, the antibody-drug conjugateprovided herein has a molar ratio of the antibody moiety to the drugmoiety that is between 1:3 and 1:5. In a specific embodiment, theantibody-drug conjugate provided herein has a molar ratio of theantibody moiety to the drug moiety that is 1:3. In another specificembodiment, the antibody-drug conjugate provided herein has a molarratio of the antibody moiety to the drug moiety that is 1:4. In anotherspecific embodiment, the antibody-drug conjugate provided herein has amolar ratio of the antibody moiety to the drug moiety that is 1:5. Inanother specific embodiment, the antibody-drug conjugate provided hereinhas a molar ratio of the antibody moiety to the drug moiety that is 1:9.

The drug moiety is conjugated to one or more chains of the antibodymoiety. In some embodiments, the drug moiety is conjugated to one chainof the antibody moiety (for example, when the antibody moiety is a scFv,or when the antibody moiety is a multi-chain antibody, such as animmunoglobulin (which is a tetramer), or antigen-binding fragmentthereof). In other embodiments, the drug moiety is conjugated to two ormore chains of the antibody moiety (when the antibody moiety is amulti-chain antibody, such as an immunoglobulin, or antigen-bindingfragment thereof). In a specific embodiment, the drug moiety isconjugated to two identical chains of an immunoglobulin, e.g., the heavychains or the light chains. In other embodiments, the drug moiety isconjugated to all chains of the antibody moiety (when the antibodymoiety is a multi-chain antibody, such as an immunoglobulin orantigen-binding fragment thereof). In some embodiments, the drug moietyis conjugated to the antibody by a method described in section 6.4infra.

In a specific embodiment, the drug moiety is conjugated to one or moresites in the constant region of an antibody. In a specific embodiment,the drug moiety is conjugated to one or more sites in the variableregion of an antibody. In a specific embodiment, the drug moiety isconjugated to one or more lysine residues in the antibody. In a specificembodiment, the drug moiety is conjugated to one or more cysteineresidues in the antibody, e.g., when MMAE or SN-38 is the drug moiety.In a particular embodiment, the drug moiety is conjugated to the Fcregion of an antibody that is an immunoglobulin. In specificembodiments, the drug moiety is a peptide or protein fused to theN-terminus or C-terminus of one or more chains of the antibody moiety(directly or via a linker that is a peptide or protein).

For example, when the antibody moiety is an scFv, the drug moiety can befused at the N- or C-terminus of the scFv (directly or via a linker thatis a peptide or protein). In a specific embodiment, when the antibodymoiety is a multi-chain antibody or antigen-binding fragment thereof,the drug moiety can be fused to one of the chains of the antibody moiety(directly or via a linker that is a peptide or protein). In the case ofan antibody that is an immunoglobulin, in a specific embodiment, bothheavy chains can be fused to the drug moiety (directly or via a linkerthat is a peptide or protein), and/or both light chains can be fused tothe drug moiety (directly or via a linker that is a peptide or protein).In such specific embodiments, provided herein are vectors (e.g.,expression vectors) comprising polynucleotides comprising a nucleotidesequence encoding a fusion protein composed of the drug moiety and theantibody moiety or a chain thereof, and the linker (if there is one)between the drug moiety and the antibody moiety, for recombinantexpression in host cells, preferably in mammalian cells. Also providedherein are ex vivo host cells comprising such vectors for recombinantlyexpressing the fusion protein composed of the drug moiety and theantibody moiety or a chain thereof, and the linker (if there is one)between the drug moiety and the antibody moiety. Characteristics of andmethods for generating such vectors and ex vivo host cells can be thesame as described below for anti-MERTK antibodies. Also provided hereinare methods of producing the fusion protein composed of the drug moietyand the antibody moiety or a chain thereof, and the linker (if there isone) between the drug moiety and the antibody moiety, comprisingculturing such an ex vivo host cell under conditions such that thepolynucleotide comprising a nucleotide sequence encoding the fusionprotein is expressed by the ex vivo host cell to produce the fusionprotein. Antibody-drug conjugates described herein may be produced by amethod known in the art or as described in Section 6 infra.

In some embodiments, the antibody-drug conjugate has the structure shownin FIG. 13A or FIG. 13B, and particularly in FIG. 13A. FIG. 13A showsthe structure of MMAE linked to an antibody (Ab) via the mc-vc-PABClinker. FIG. 13B shows the structure of SN-38 linked to an antibody (Ab)via the CL2 or CL2A linker. If “AA” in FIG. 13B is phenylalanine-lysine,then FIG. 13B shows an antibody linked to SN-38 via the CL2 linker(which comprises a Cathepsin B cleavable site). If “AA” in FIG. 13B islysine, then FIG. 13B shows an antibody linked to SN-38 via the CL2Alinker (which does not comprise a Cathepsin B cleavable site).

In a specific embodiment, the antibody portion of the antibody-drugconjugate comprises the variable regions of antibody z10. In a specificembodiment, an antibody-drug conjugate provided herein comprises anantibody moiety comprising a heavy chain variable region (VH) and alight chain variable region (VL), wherein the VH comprises the aminoacid sequence of sequence of SEQ ID NO: 105 and the VL comprises theamino acid sequence of SEQ ID NO: 106, wherein the antibody moiety isconjugated to MMAE via the mc-vc-PABC linker. In another specificembodiment, an antibody-drug conjugate provided herein comprises anantibody moiety comprising a heavy chain variable region (VH) and alight chain variable region (VL), wherein the VH comprises the aminoacid sequence of sequence of SEQ ID NO: 105 and the VL comprises theamino acid sequence of SEQ ID NO: 106, wherein the antibody moiety isconjugated to SN-38 via the CL2A linker.

In an alternative embodiment of the antibody-drug conjugates describedthroughout this disclosure, the drug moiety is not necessarily acytotoxic agent. For example, the drug moiety can be any drug known inthe art suitable for use, and may include but not be limited to thosedescribed in section 5.2.3 infra, or, e.g., for purposes of diagnosis ormonitoring of disease, can be an imaging agent.

In a preferred embodiment of the antibody-drug conjugates describedthroughout this disclosure, the drug moiety is conjugated to theantibody (also referred to herein as the “antibody moiety”) via alinker.

5.2.1. Functional Characteristics of the Antibody-Drug Conjugates

In certain embodiments, an antibody-drug conjugate described herein,binds to MERTK (e.g. human MERTK) with a K_(D) of about 8 nM, 7 nM, 6nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.75nM, 0.5 nM, 0.25 nM, 0.17 nM, 0.1 nM, 0.081 nM or 0.021 nM. In someaspects, an antibody-drug conjugate provided herein decreases expressionlevels of human MERTK on cancer cells (e.g. SKMEL5 melanoma cells) bymore than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80% or 85% as assessed by an assay known to one of skill inthe art or described herein, e.g., as described in section 6 infra. Inspecific embodiments, an antibody-drug conjugate provided herein bindsto MERTK (e.g., binds to human MERTK but not human Axl, human Tyro3, ormurine MERTK), as assessed by methods described herein or known in theart. In certain embodiments, an antibody-drug conjugate provided hereincomprises an antibody moiety wherein the antibody-drug conjugate bindsto MERTK with greater affinity than the unconjugated antibody moiety.

In certain embodiments, an antibody-drug conjugate provided hereincauses cell death of MERTK (e.g., human MERTK)-expressing cells (e.g.,M2 macrophages, cancer cells, or both) in vitro. In some specificembodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, or 85% of MERTK-expressing cells cultured for aperiod of time in the presence of an antibody-drug conjugate describedherein are dead at the end of the period of time, as assessed by methodsknown to one of skill in the art for measuring cell death in vitro. Inother specific embodiments, the percentage of MERTK-expressing cells(e.g., M2 macrophages, cancer cells, or both) that undergo cell death isat least 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold,100-fold, 200-fold, 500-fold, or 1000-fold higher when cultured for aperiod of time in the presence of an antibody-drug conjugate describedherein, relative to when cultured without the antibody-drug conjugate(e.g., cultured without any antibody-drug conjugate or cultured with anantibody-drug conjugate comprising an unrelated antibody (e.g., anantibody that does not immunospecifically bind to MERTK (e.g., humanMERTK)) or cultured with an unconjugated anti-MERTK antibody orunconjugated anti-MERTK antigen-binding fragment described herein). Theperiod of time can be, for example, about 1 hr, 2 hrs, 4 hrs, 8 hrs, 12hrs, 16 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or a week.

In some embodiments, an antibody-drug conjugate provided herein does notaffect viability of MERTK-expressing macrophages as assessed by methodsknown to one of skill in the art for measuring cell death in vitro or amethod described herein, e.g., in section 6 infra. In some embodiments,an antibody-drug conjugate provided herein does not affect viability ofM1 macrophages as assessed by methods known to one of skill in the artfor measuring cell death in vitro or a method described herein, e.g., insection 6 infra. In some embodiments, an antibody-drug conjugateprovided herein does not affect viability of M2 macrophages as assessedby methods known to one of skill in the art for measuring cell death invitro or a method described herein, e.g., in section 6 infra.

In certain embodiments, an antibody-drug conjugate provided herein,induces degradation of MERTK on MERTK expressing cells, such as, e.g.,cancer cells (e.g., melanoma cells). In specific embodiments, anantibody-drug conjugate provided herein reduces the level of human MERTKon the surface of human MERTK expressing cells, such as, e.g., cancercells (e.g., melanoma cells) by at least 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% in an assay describedherein or known to one of skill in the art. In certain embodiments, anantibody-drug conjugate provided herein induces greater degradation ofMERTK on MERTK expressing cells than the unconjugated antibody moiety,as assessed in an assay described herein or known in the art.

In certain embodiments, an antibody-drug conjugate provided hereininduces internalization of MERTK on MERTK expressing cells, such as,e.g., cancer cells (e.g., melanoma cells). In specific embodiments, anantibody-drug conjugate provided herein increases the internalization ofhuman MERTK by human MERTK expressing cells, such as, e.g., cancer cells(e.g., melanoma cells), by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% in an assay describedherein or known to one of skill in the art. In certain embodiments, anantibody-drug conjugate provided herein comprises an antibody moiety andthe antibody-drug induces greater internalization of MERTK expressingcells than the unconjugated antibody moiety as assessed by an assaydescribed herein or known to one of skill in the art.

In certain embodiments, an antibody-drug conjugate provided hereininhibits tumor progression. The inhibition of tumor progression by atleast 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55, 60%, 65%, 70%,75%, 80% or 85%. Tumor progression can be assessed by methods known toone of skill in the art. The tumor progression can be relative to thecancer status without treatment with any antibody, treated with anunrelated antibody (e.g., an antibody that does not specifically bind tohuman MERTK), treated with SN-38, treated with MMAE, treated with theunconjugated antibody moiety of the antibody-drug conjugate. In aspecific embodiment, an anti-MERTK antibody or an antigen-binding regionthereof has one, two, three or more of the characteristics of anantibody described in Section 6, infra.

In specific embodiments, an antibody-drug conjugate described hereininhibits angiogenesis within tumors. In some embodiments, the inhibitionof angiogenesis is by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%. In a specific embodiment, theinhibition of angiogenesis is at least 50%, 55%, 60%, 65%, or 70%.Inhibition of angiogenesis can be assessed by methods described hereinand/or known to one of skill in the art. The inhibition can be relativeto the level of angiogenesis without the antibody-drug conjugate (e.g.,cultured without any antibody-drug conjugate or cultured with anantibody-drug conjugate comprising an unrelated antibody (e.g., anantibody that does not specifically bind to MERTK (e.g., human MERTK))or cultured with an unconjugated anti-MERTK antibody or unconjugatedanti-MERTK antigen-binding fragment described herein).

In certain embodiments, an antibody-drug conjugate described hereininhibits tumor (e.g., human breast cancer tumor) progression. Theinhibition of tumor progression by at least 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%. Tumorprogression can be assessed by methods known to one of skill in the art.The tumor progression can be relative to the cancer status without theantibody-drug conjugate (e.g., cultured without any antibody-drugconjugate or cultured with an antibody-drug conjugate comprising anunrelated antibody (e.g., an antibody that does not specifically bind toMERTK (e.g., human MERTK)) or cultured with an unconjugated anti-MERTKantibody or unconjugated anti-MERTK antigen-binding fragment describedherein). In a specific embodiment, the assay that is used to assesstumor progression is a murine tumor transplantation model.

5.2.2. The Antibody Moiety

An antibody-drug conjugate provided herein may comprise any anti-MERTKantibody provided herein. See, e.g., Section 5.1.1 for examples ofantibody moieties of the antibody-drug conjugates provided herein. In aspecific embodiment, the antibody moiety is an immunoglobulin, e.g., animmunoglobulin comprising a human constant domain, or a humanizedimmunoglobulin. In another specific embodiment, the antibody moiety is ascFv, e.g., comprising the VH and VL domains of an antibody describedherein in Section 5.1.1

In some embodiments, the antibody-drug conjugate provided hereincomprises a drug moiety conjugated optionally via a linker to anantibody moiety comprising a VH disclosed herein, e.g., a VH describedin Section 5.1.1. In some embodiments, the antibody-drug conjugateprovided herein comprises a drug moiety conjugated optionally via alinker to an antibody moiety comprising a VL disclosed herein, e.g., aVL described in Section 5.1.1. In some embodiments, the antibody-drugconjugate provided herein comprises a drug moiety conjugated optionallyvia a linker to an antibody moiety comprising a VH and a VL of anantibody disclosed herein, e.g., as described in Section 5.1.1.

In some embodiments, the antibody-drug conjugate provided hereincomprises a drug moiety conjugated optionally via a linker to anantibody moiety comprising a VH comprising the amino acid sequence ofSEQ ID NO: 105. In some embodiments, the antibody-drug conjugateprovided herein comprises a drug moiety conjugated optionally via alinker to an antibody moiety comprising a VL comprising the amino acidsequence of SEQ ID NO: 106. In some embodiments, the antibody-drugconjugate provided herein comprises a drug moiety conjugated optionallyvia a linker to an antibody moiety comprising (i) a VH comprising theamino acid sequence of SEQ ID NO: 105 and (ii) a VL comprising the aminoacid sequence of SEQ ID NO: 106.

In some embodiments, an antibody-drug conjugate provided hereincomprises MMAE conjugated via a linker to an antibody moiety comprisinga VH, wherein the VH comprises the amino acid sequence of SEQ ID NO:105. In some embodiments, an antibody-drug conjugate provided hereincomprises MMAE conjugated via a linker to an antibody moiety comprisinga VL, wherein the VL comprises the amino acid sequence of SEQ ID NO:106. In some embodiments, an antibody-drug conjugate provided hereincomprises SN-38 conjugated via a linker to an antibody moiety comprisinga VH, wherein the VH comprises the amino acid sequence of SEQ ID NO:105. In some embodiments, an antibody-drug conjugate provided hereincomprises SN-38 conjugated via a linker to an antibody moiety comprisinga VL, wherein the VL comprises the amino acid sequence of SEQ ID NO:106.

In a specific embodiment, an antibody-drug conjugate provided hereincomprises MMAE conjugated via a linker to an antibody moiety comprisinga VH and a VL, wherein the VH comprises the amino acid sequence of SEQID NO: 105, and the VL comprises the amino acid sequence of SEQ ID NO:106. In another specific embodiment, an antibody-drug conjugate providedherein comprises SN-38 conjugated via a linker to an antibody moietycomprising a VH and a VL, wherein the VH comprises the amino acidsequence of SEQ ID NO: 105, and the VL comprises the amino acid sequenceof SEQ ID NO: 106.

In some embodiments, the antibody-drug conjugate provided hereincomprises a drug moiety conjugated via a linker to an antibody moietycomprising a heavy chain provided herein, e.g., a heavy chain describedin Section 5.1.1. In some embodiments, the antibody-drug conjugateprovided herein comprises MMAE conjugated via a linker to an antibodymoiety comprising a heavy chain provided herein, e.g., a heavy chaindescribed in Section 5.1.1. In some embodiments, the antibody-drugconjugate provided herein comprises SN-38 conjugated via a linker to anantibody moiety comprising a heavy chain provided herein, e.g., a heavychain described in Section 5.1.1. In some embodiments, the antibody-drugconjugate provided herein comprises a drug moiety conjugated via alinker to an antibody moiety comprising a light chain provided herein,e.g., a light chain described in Section 5.1.1. In some embodiments, theantibody-drug conjugate provided herein comprises MMAE conjugated via alinker to an antibody moiety comprising a light chain provided herein,e.g., a light chain described in Section 5.1.1. In some embodiments, theantibody-drug conjugate provided herein comprises SN-38 conjugated via alinker to an antibody moiety comprising a light chain provided herein,e.g., a light chain described in Section 5.1.1. In some embodiments, theantibody-drug conjugate provided herein comprises a drug moietyconjugated via a linker to an antibody moiety comprising (i) a heavychain provided herein and (ii) a light chain provided herein, e.g., aheavy chain and a light chain described in Section 5.1.1. In someembodiments, the antibody-drug conjugate provided herein comprises MMAEconjugated via a linker to an antibody moiety comprising (i) a heavychain provided herein and (ii) a light chain provided herein, e.g., aheavy chain and a light chain described in Section 5.1.1. In someembodiments, the antibody-drug conjugate provided herein comprises SN-38conjugated via a linker to an antibody moiety comprising (i) a heavychain provided herein and (ii) a light chain provided herein, e.g., aheavy chain and a light chain described in Section 5.1.1.

In some embodiments, the antibody-drug conjugate provided hereincomprises a drug moiety conjugated via a linker to an antibody moietycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 112. In some specific embodiments, the antibody-drug conjugateprovided herein comprises MMAE conjugated via a linker to an antibodymoiety comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 112. In some specific embodiments, the antibody-drugconjugate provided herein comprises SN-38 conjugated via a linker to anantibody moiety comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 112. In some embodiments, the antibody-drugconjugate provided herein comprises a drug moiety conjugated via alinker to an antibody moiety comprising a light chain comprising theamino acid of SEQ ID NO: 113. In some specific embodiments, theantibody-drug conjugate provided herein comprises MMAE conjugated via alinker to a light chain comprising the amino acid of SEQ ID NO: 113. Insome specific embodiments, the antibody-drug conjugate provided hereincomprises SN-38 conjugated via a linker to a light chain comprising theamino acid of SEQ ID NO: 113. In some embodiments, the antibody-drugconjugate provided herein comprises a drug moiety conjugated via alinker to an antibody moiety comprising (i) a heavy chain comprising theamino acid sequence of SEQ ID NO: 112 and (ii) a light chain comprisingthe amino acid of SEQ ID NO: 113. In a specific embodiment, theantibody-drug conjugate provided herein comprises MMAE conjugated via alinker to an antibody moiety comprising (i) a heavy chain comprising theamino acid sequence of SEQ ID NO: 112 and (ii) a light chain comprisingthe amino acid of SEQ ID NO: 113. In another specific embodiment, theantibody-drug conjugate provided herein comprises SN-38 conjugated via alinker to an antibody moiety comprising (i) a heavy chain comprising theamino acid sequence of SEQ ID NO: 112 and (ii) a light chain comprisingthe amino acid of SEQ ID NO: 113.

5.2.3. The Drug Moiety and the Linker

The cytotoxic agent used in antibody-drug conjugates is also oftencalled a payload or warhead. Most of the cytotoxic agents used inantibody-drug conjugates target DNA or microtubules and have highpotency of cytotoxicity (with an IC₅₀ range of approximately 10⁻¹⁰-10⁻¹²M) (see Beck A et al., (2017) Nat Rev Drug Discov 16: 315-337). However,cytotoxic agents that are not as potent may also be used. The cytotoxicagent can be a small molecule, a nucleotide, a peptide, or anon-antibody protein. In a specific embodiment, the cytotoxic agent is asmall molecule. In another specific embodiment, the cytotoxic agent is anon-antibody protein. Non-limiting exemplary cytotoxic agents that canbe used in the antibody-drug conjugates according to the invention aredescribed in Beck A et al., (2017) Nat Rev Drug Discov 16: 315-337;Peters C and Brown S, (2015) Biosci Rep 35: art:e00225; McCombs J R andOwen S C (2015) The AAPS Journal 17: 339-351; Jackson D Y (2016) OrgProcess Res Dev 20: 852-866; and Olivier K J and Hurvitz S A ed., (2016)Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical,Wiley.

In certain embodiments, the cytotoxic agent is one of the agents listedin Table 30 or 31. In specific embodiments, the cytotoxic agent used inthe antibody-drug conjugate is an auristatin (such as monomethylauristatin E (MMAE), monomethyl auristatin F (MMAF), Aur0101,PF06380101, Auristatin W, or auristatin F) or derivative thereof, amaytansinoid (such as DM1 or DM4), a pyrrolobenzodiazepine (PBD) (suchas SGD1882 or SG3199), an indolinobenzodiazepine (such as DGN462 orDGN549), a calicheamicin (ozogamicin) (such as CM1), a camptothecinanalogue (such as SN-38, DX-895 if, or DX-895 if derivative), aduocarmycin (such as seco-duocarmycin-hydroxy-benzamide-azaindole(seco-DUBA), minor groove-binding alkylating agent (MGBA), or MED-2460),a tubulin inhibitor (such as cryptophycin), a tubulysin or tubulysinanalogue (such as AZ13599185), amberstatin269, doxorubicin, anantibiotic (such as rifalogue), an anthracycline (such as PNU-159682), amicrotubule inhibitor (such as rhizoxin), a spliceostatin, or athailanstatin. In a specific embodiment, the cytotoxic agent used in theantibody-drug conjugate is MMAE or MMAF. In a specific embodiment, thecytotoxic agent used in the antibody-drug conjugate is MMAE (see, e.g.,FIG. 13A). In another specific embodiment, the cytotoxic agent used inthe antibody-drug conjugate is DM1 or DM4. In another specificembodiment, the cytotoxic agent used in the antibody-drug conjugate isSN-38. In another specific embodiment, the cytotoxic agent used in theantibody-drug conjugate is SN-38 as shown in FIG. 13B.

TABLE 30 Examples of Drug Moieties Alkylating agents BusulfanChlorambucil Dacarbazine Procarbazine Ifosfamide Altretaminehexamethylmelamine estramustine phosphate Thiotepa MechlorethamineDacarbazine Streptozocin Lomustine Temozolomide cyclophosphamideSemustine Platinum agents Spiroplatin lobaplatin (Aeterna) Tetraplatinsatraplatin (Johnson Matthey) Ormaplatin BBR-3464 (Hoffmann-La Roche)Iproplatin SM-11355 (Sumitomo) ZD-0473 (AnorMED) AP-5280 (Access)Oxaliplatin Cisplatin Carboplatin Antimetabolites AzacytidineTrimetrexate Floxuridine Deoxycoformycin 2-chlorodeoxyadenosinePentostatin 6-mercaptopurine Hydroxyurea 6-thioguanine decitabine(SuperGen) Cytarabine clofarabine (Bioenvision) 2-fluorodeoxy cytidineirofulven (MGI Pharma) Methotrexate DMDC (Hoffmann-La Roche) tomudexethynylcytidine (Taiho) Fludarabine Gemcitabine Raltitrexed CapecitabineTopoisomerase Amsacrine exatecan mesylate (Daiichi) InhibitorsEpirubicin quinamed (ChemGenex) Etoposide gimatecan (Sigma-Tau)teniposide or mitoxantrone diflomotecan (Beaufour-Ipsen)7-ethyl-10-hydroxy- TAS-103 (Taiho) camptothecin dexrazoxanet(TopoTarget) elsamitrucin (Spectrum) pixantrone (Novuspharma) J-107088(Merck & Co) rebeccamycin analogue BNP-1350 (BioNumerik) (Exelixis)BBR-3576 (Novuspharma) CKD-602 (Chong Kun Dang) rubitecan (SuperGen)KW-2170 (Kyowa Hakko) irinotecan (CPT-11) hydroxycamptothecin (SN-38)Topotecan Antitumor Valrubicin Azonafide antibiotics TherarubicinAnthrapyrazole Idarubicin Oxantrazole Rubidazone Losoxantrone PlicamycinMEN-10755 (Menarini) Porfiromycin GPX-100 (Gem Pharmaceuticals)mitoxantrone (novantrone) Epirubicin Amonafide Mitoxantrone DoxorubicinAntimitotic Colchicine E7010 (Abbott) Agents Vinblastine PG-TXL (CellTherapeutics) Vindesine IDN 5109 (Bayer) dolastatin 10 (NCI) A 105972(Abbott) rhizoxin (Fujisawa) A 204197 (Abbott) mivobulin(Warner-Lambert) LU 223651 (BASF) cemadotin (BASF) D 24851 (ASTAMedica)RPR 109881A (Aventis) ER-86526 (Eisai) TXD 258 (Aventis) combretastatinA4 (BMS) epothilone B (Novartis) isohomohalichondrin-B (PharmaMar) T900607 (Tularik) ZD 6126 (AstraZeneca) T 138067 (Tularik) AZ 10992(Asahi) cryptophycin 52 (Eli Lilly) IDN-5109 (Indena) vinflunine (Fabre)AVLB (Prescient NeuroPharma) auristatin PE (Teikoku azaepothilone B(BMS) Hormone) BMS 247550 (BMS) BNP-7787 (BioNumerik) BMS 184476 (BMS)CA-4 prodrug (OXiGENE) BMS 188797 (BMS) dolastatin-10 (NIH) taxoprexin(Protarga) CA-4 (OXiGENE) SB 408075 Docetaxel (GlaxoSmithKline)Vinorelbine Vincristine Trichostatin A Paclitaxel Aromataseaminoglutethimide YM-511 (Yamanouchi) inhibitors atamestane(BioMedicines) Formestane Letrozole Exemestane Anastrazole Thymidylatepemetrexed (Eli Lilly) nolatrexed (Eximias) synthase inhibitors ZD-9331(BTG) CoFactor ™ (BioKeys) DNA antagonists trabectedin (PharmaMar)edotreotide (Novartis) glufosfamide (Baxter mafosfamide (BaxterInternational) International) albumin + 32P (Isotope apaziquone(Spectrum Solutions) Pharmaceuticals) thymectacin (NewBiotics) O6 benzylguanine (Paligent) Farnesyltransferase arglabin (NuOncology Labs)tipifarnib (Johnson & Johnson) Inhibitors lonafarnib (Schering-Plough)perillyl alcohol (DOR BioPharma) BAY-43-9006 (Bayer) Pump inhibitorsCBT-1 (CBA Pharma) zosuquidar trihydrochloride (Eli Lilly) tariquidar(Xenova) biricodar dicitrate (Vertex) MS-209 (Schering AG) Histonetacedinaline (Pfizer) pivaloyloxymethyl butyrate (Titan)acetyltransferase SAHA (Aton Pharma) depsipeptide (Fujisawa) InhibitorsMS-275 (Schering AG) Metalloproteinase Neovastat (Aeterna CMT-3(CollaGenex) Inhibitors Laboratories) marimastat (British Biotech)BMS-275291 (Celltech) Ribonucleoside gallium maltolate (Titan)tezacitabine (Aventis) reductase inhibitors triapine (Vion) didox(Molecules for Health) TNF alpha virulizin (Lorus Therapeutics) revimid(Celgene) agonists/antagonists CDC-394 (Celgene) YM-598 (Yamanouchi)Endothelin A atrasentan (Abbott) alitretinoin (Ligand) receptorantagonist ZD-4054 (AstraZeneca) ISF-154 (Tragen) Retinoic acidfenretinide (Johnson & β-alethine (Dovetail) receptor agonists Johnson)LGD-1550 (Ligand) CLL therapy (Vasogen) Immunomodulators InterferonMAGE-A3 (GSK) IRX-2 (Immuno-Rx) CM-10 (cCam Biotherapeutics) PEP-005(Peplin Biotech) AMP-224 (GSK) abatacept (BMS) MPDL3280A (Genentech)Lag-3 GITR TGF-beta OX40 RGX-104 RGX-202 Hormonal and Estrogensdexamethasone antihormonal conjugated estrogens Prednisone agentsethinyl estradiol methylprednisolone Chlortrianisen prednisoloneIdenestrol aminoglutethimide hydroxyprogesterone caproate Leuprolidemedroxyprogesterone Octreotide Testosterone Mitotane testosteronepropionate; P-04 (Novogen) fluoxymesterone methyltestosterone2-methoxyestradiol (EntreMed) diethylstilbestrol arzoxifene (Eli Lilly)Megestrol Tamoxifen Bicalutamide Toremofine Flutamide GoserelinNilutamide Leuporelin bicalutamide Photodynamic talaporfin (LightSciences) Pd-bacteriopheophorbide (Yeda) Agents Theralux(Theratechnologies) lutetium texaphyrin (Pharmacyclics) motexafingadolinium Hypericin (Pharmacyclics) Kinase Inhibitors imatinib(Novartis) EKB-569 (Wyeth) leflunomide kahalide F (PharmaMar)(Sugen/Pharmacia) ZD1839 (AstraZeneca) CEP-701 (Cephalon) erlotinib(Oncogene Science) CEP-751 (Cephalon) canertinib (Pfizer) MLN518(Millenium) squalamine (Genaera) PKC412 (Novartis) SU5416 (Pharmacia)Phenoxodiol (Novogen) SU6668 (Pharmacia) C225 (ImClone) ZD4190(AstraZeneca) rhu-Mab (Genentech) ZD6474 (AstraZeneca) MDX-H210(Medarex) vatalanib (Novartis) 2C4 (Genentech) PKI166 (Novartis) MDX-447(Medarex) GW2016 (GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth)IMC-1C11 (ImClone) trastuzumab (Genentech) Tyrphostins OSI-774(Tarceva ™) Gefitinib (Iressa) CI-1033 (Pfizer) PTK787 (Novartis)SU11248 (Pharmacia) EMD 72000 (Merck) RH3 (York Medical) EmodinGenistein Radicinol Radicinol Vemurafenib (B-Raf enzyme inhibitor,Daiichi Sankyo) Met-MAb (Roche) trametinib (GSK)

TABLE 31 Additional examples of drug moieties. SR-27897 (CCK Ainhibitor, CDA-II (apoptosis promotor, triacetyluridine (uridineSanofi-Synthelabo) Everlife) prodrug, Wellstat) tocladesine (cyclic AMPSDX-101 (apoptosis SN-4071 (sarcoma agent, agonist, Ribapharm) promotor,Salmedix) Signature BioScience) alvocidib (CDK inhibitor, CarmustineTransMID-107 ™ Aventis) (immunotoxin, KS Biomedix) CV-247 (COX-2inhibitor, Mitoxantrone PCK-3145 (apoptosis Ivy Medical) promotor,Procyon) P54 (COX-2 inhibitor, Bleomycin doranidazole (apoptosisPhytopharm) promotor, Pola) CapCell ™ (CYP450 Absinthin CHS-828(cytotoxic agent, stimulant, Bavarian Nordic) Leo) GCS-100 (gal3antagonist, Chrysophanic acid trans-retinoic acid GlycoGenesys)(differentiator, NIH) G17DT immunogen (gastrin Cesium oxides MX6(apoptosis promotor, inhibitor, Aphton) MAXIA) efaproxiral (oxygenator,BRAF inhibitors, apomine (apoptosis promotor, Allos Therapeutics) ILEXOncology) PI-88 (heparanase inhibitor, PDL1 inhibitors urocidin(apoptosis promotor, Progen) Bioniche) tesmilifene (histamine MEKinhibitors Ro-31-7453 (apoptosis antagonist, YM BioSciences) promotor,La Roche) histamine (histamine H2 angiogenesis inhibitors brostallicin(apoptosis receptor agonist, Maxim) promotor, Pharmacia) tiazofurin(IMPDH inhibitor, dabrafenib β-lapachone Ribapharm) cilengitide(integrin ceflatonin (apoptosis Gelonin antagonist, Merck KGaA)promotor, ChemGenex) SR-31747 (IL-1 antagonist, BCX-1777 (PNP inhibitor,Cafestol Sanofi-Synthelabo) BioCryst) CCI-779 (mTOR kinase ranpirnase(ribonuclease Kahweol inhibitor, Wyeth) stimulant, Alfacell) exisulind(PDE V inhibitor, galarubicin (RNA synthesis caffeic acid Cell Pathways)inhibitor, Dong-A) CP-461 (PDE V inhibitor, tirapazamine (reducingagent, Tyrphostin AG Cell Pathways) SRI International) AG-2037 (GARTinhibitor, N-acetylcysteine (reducing PD-1 inhibitors Pfizer) agent,Zambon) WX-UK1 (plasminogen R-flurbiprofen (NF-kappaB CTLA-4 inhibitorsactivator inhibitor, Wilex) inhibitor, Encore) PBI-1402 (PMN stimulant,3CPA (NF-kappaB inhibitor, Sorafenib ProMetic LifeSciences) ActiveBiotech) bortezomib (proteasome seocalcitol (vitamin D BRAF inhibitorsinhibitor, Millennium) receptor agonist, Leo) SRL-172 (T cell stimulant,131-I-TM-601 (DNA indisulam (p53 stimulant, SR Pharma) antagonist,TransMolecular) Eisai) TLK-286 (glutathione S eflornithine (ODCinhibitor, aplidine (PPT inhibitor, transferase inhibitor, Telik) ILEXOncology) PharmaMar) PT-100 (growth factor minodronic acid (osteoclastgemtuzumab (CD33 agonist, Point Therapeutics) inhibitor, Yamanouchi)antibody, Wyeth Ayerst) midostaurin (PKC inhibitor, PG2 (hematopoiesisNovartis) enhancer, Pharmagenesis) bryostatin-1 (PKC stimulant,Immunol ™ (triclosan oral GPC Biotech) rinse, Endo)

As used herein, the term “small molecule” refers to an organic orinorganic compound (which can be, for example, a heteroorganic ororganometallic compound) having a molecular weight of less than about10,000 grams per mole. In specific embodiments, the small molecule has amolecular weight of less than about 5,000 grams per mole, or less thanabout 2,000 grams per mole, or less than about 1,000 grams per mole, orless than about 500 grams per mole, or less than about 100 grams permole.

In some embodiments, the cytotoxic agent is conjugated directly to theantibody moiety. In other embodiments, the cytotoxic agent is conjugatedto the antibody moiety via a linker. Appropriate linkers to be usedaccording to the invention preferably are stable in the blood stream tolimit off-target toxicity and labile at the cancer site to allow forrelease of the cytotoxic agent (see Peters C and Brown S, (2015) BiosciRep 35: art:e00225). Non-limiting exemplary linkers that can be usedaccording to the invention are described in Beck A et al., (2017) NatRev Drug Discov 16: 315-337; Peters C and Brown S, (2015) Biosci Rep 35:art:e00225; McCombs J R and Owen S C (2015) The AAPS Journal 17:339-351; Jackson D Y (2016) Org Process Res Dev 20: 852-866; and OlivierK J and Hurvitz S A ed., (2016) Antibody-Drug Conjugates: Fundamentals,Drug Development, and Clinical, Wiley. In some embodiments, the linkeris a cleavable linker, for example, having a motif sensitive to alysosomal protease (for example, cathepsin B), a motif sensitive to anacidic pH (for example, hydrazone), or a motif containing a disulfidebridge that can be reduced by glutathione. In other embodiments, thelinker is a non-cleavable linker. By way of example but not limitation,the linker can be a small molecule, a nucleotide, a peptide, or anon-antibody protein. In a specific embodiment, the linker is a smallmolecule. In a specific embodiment, the linker used in the antibody-drugconjugate is cathepsin B, hydrazone,succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),maleimidocaproic acid (mc), valine-citrulline (vc),N-hydroxysuccinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB),N-hydroxysuccinimidyl 4-(2-pyridydithio)butanoate (SPDB), N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP), valine-alanine (va), polyethyleneglycol 8-valine-citrulline (PEG8-va), mb-vc, CL2A, cleavable vc-basedlinker, or fleximer polymer linker. In a specific embodiment, a linkerselected for use in an antibody drug conjugate described herein does notinterfere with the antibody moiety binding to its antigen, the cytotoxicagent, or both. In specific embodiments, a linker selected for use in anantibody-drug conjugate described herein ismaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl(mc-vc-PABC). In other specific embodiments, a linker selected for usein an antibody-drug conjugate described herein is CL2. In other specificembodiments, a linker selected for use in an antibody-drug conjugatedescribed herein is CL2A. In some specific embodiments, a linkerselected for use in an antibody drug conjugate described herein is thelinker shown in FIG. 13A. In other specific embodiments, a linkerselected for use in an antibody-drug conjugate described herein is thelinker shown in FIG. 13B where AA is lysine. In other specificembodiments, a linker selected for use in an antibody-drug conjugatedescribed herein is the linker shown in FIG. 13B where AA isphenylalanine-lysine. In a specific embodiment, the antibody-drugconjugate comprises a linker and the linker ismaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (also knownas “mc-vc-PABC”) (for the structure of mc-vc-PABC; see FIG. 13A). Inanother specific embodiment, the antibody-drug conjugate comprises alinker and the linker is CL2 (for the structure of CL2, see FIG. 13B andCardillo et al. 2011, Clin Cancer Res; 17(10); 3157-69). In anotherspecific embodiment, the antibody-drug conjugate comprises the linkerand the linker is CL2A (for the structure of CL2A, see FIG. 13B andGoldberg et al. 2018, Oncotarget; 9(48); 28989-29006, and Cardillo etal. 2011, Clin Cancer Res; 17(10); 3157-69).

In some embodiments, a linker selected for use in an antibody-drugconjugate described herein comprises one or more cleavage sites. Inspecific embodiments, a linker selected for use in an antibody-drugconjugate described herein contains a Cathepsin B protease site. Inother specific embodiments, a linker selected for use in anantibody-drug conjugate described herein contains a Cathepsin B proteasesite and a pH-dependent cleavage site.

Non-limiting exemplary linker-drug moiety pairs that can be used inantibody-drug conjugates described herein are described in Beck A etal., (2017) Nat Rev Drug Discov 16: 315-337; Peters C and Brown S,(2015) Biosci Rep 35: art:e00225; McCombs J R and Owen S C (2015) TheAAPS Journal 17: 339-351; Jackson D Y (2016) Org Process Res Dev 20:852-866; and Olivier K J and Hurvitz S A ed., (2016) Antibody-DrugConjugates: Fundamentals, Drug Development, and Clinical, Wiley. Inspecific embodiments, the linker-drug moiety pair in the antibody-drugconjugate is vc-MMAE, mc-MMAF, SMCC-DM1, sulfo-SPDB-DM4, SPDB-DM4,SPP-DM1, va-SGD1882, polyethylene glycol 8 (PEG8)-va-SG3199,sulfo-SPDB-DGN462, hydrazone-CM1, vc-seco-DUBA, mb-vc-MGBA, CL2A-SN-38,peptide linker with DX-8951 derivative, hydrazone-doxorubicin, cleavablevc-based linker with Aur0101, vc-PF06380101, fleximer polymer linkerwith auristatin F, cleavable linker-tubulin inhibitor, or vc-rifalogue.In specific embodiments, the linker-drug moiety pair in theantibody-drug conjugate is mc-vc-PABC-MMAE (as shown in FIG. 13A). Inother specific embodiments, the linker-drug moiety pair in theantibody-drug conjugate is CL2-SN-38 (as shown in FIG. 13B where AA isphenylalanine-lysine). In other specific embodiments, the linker-drugmoiety pair in the antibody-drug conjugate is CL2A-SN-38 (as shown inFIG. 13B where AA is lysine).

In a specific embodiment, the drug moiety of an antibody-drug conjugatedescribed herein does not interfere with the antibody moiety binding toits antigen (e.g., MERTK (e.g., human MERTK)).

5.3. Antibody Production

5.3.1. Producing and Screening Antibodies

In another aspect, provided herein are methods of producing anti-MERTKantibodies or antigen-binding fragments thereof described herein, thatspecifically bind to MERTK (e.g., human MERTK).

The antibodies or antigen-binding fragments thereof described herein canbe produced by any method known in the art for the synthesis ofantibodies, for example, by chemical synthesis or by recombinantexpression techniques. The methods described herein employs, unlessotherwise indicated, conventional techniques in molecular biology,microbiology, genetic analysis, recombinant DNA, organic chemistry,biochemistry, PCR, oligonucleotide synthesis and modification, nucleicacid hybridization, and related fields within the skill of the art.These techniques are described, for example, in the references citedherein and are fully explained in the literature. See, e.g., Maniatis Tet al., (1982) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press;Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel F M etal., Current Protocols in Molecular Biology, John Wiley & Sons (1987 andannual updates); Current Protocols in Immunology, John Wiley & Sons(1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: APractical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotidesand Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.)(1999) Genome Analysis: A Laboratory Manual, Cold Spring HarborLaboratory Press.

In a specific embodiment, an antibody described herein is an antibody(e.g., recombinant antibody) prepared, expressed, created or isolated byany means that involves creation, e.g., via synthesis, geneticengineering of DNA sequences. In certain embodiments, such antibodycomprises sequences that are encoded by DNA sequences that do notnaturally exist within the antibody germline repertoire of an animal ormammal (e.g., human) in vivo. In a specific embodiment, an antibodydescribed herein is made by a method comprising using human MERTK (SEQID NO: 131 or the extracellular domain thereof (SEQ ID NO: 132) as animmunogen.

In a certain aspect, provided herein is a method of making an anti-MERTKantibody or antigen-binding fragment thereof, which specifically bindsto MERTK (e.g., human MERTK), comprising culturing a cell or host celldescribed herein. In a certain aspect, provided herein is a method ofmaking an anti-MERTK antibody or antigen-binding fragment thereof, whichspecifically binds to MERTK (e.g., human MERTK), comprising expressing(e.g., recombinantly expressing) the antibody or antigen-bindingfragment thereof using a cell or host cell described herein (e.g., acell or a host cell comprising polynucleotides encoding an antibodydescribed herein). In a particular embodiment, the cell is an isolatedcell. In a particular embodiment, the exogenous polynucleotides havebeen introduced into the cell. In a particular embodiment, the methodfurther comprises the step of purifying the antibody or antigen-bindingfragment thereof obtained from the cell or host cell.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York).

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. Monoclonal antibodiescan be prepared using a wide variety of techniques known in the artincluding the use of hybridoma, recombinant, and phage displaytechnologies, or a combination thereof. For example, monoclonalantibodies can be produced recombinantly from host cells exogenouslyexpressing an antibody described herein or a fragment thereof, forexample, a light chain and/or heavy chain of such antibody. Methods forthe preparation of clonal cell lines and of monoclonal antibodiesexpressed thereby are well known in the art (see, for example, Chapter11 in Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel F Met al., supra). For example, monoclonal antibodies can be produced usinghybridoma techniques including those known in the art and taught, forexample, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2nd ed. 1988); Hammerling G J et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y.,1981); and Kohler G & Milstein C (1975) Nature 256: 495. Methods forproducing and screening for specific antibodies using hybridomatechnology are routine and well known in the art. In some embodiments,mice (or other animals, such as rats, monkeys, donkeys, pigs, sheep,hamster, or dogs) can be immunized with an antigen (e.g., human) andonce an immune response is detected, e.g., antibodies specific for theantigen are detected in the mouse serum, the mouse spleen is harvestedand splenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example cells from cellline SP2/0 available from the American Type Culture Collection (ATCC®)(Manassas, Va.), to form hybridomas. Hybridomas are selected and clonedby limited dilution.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against MERTK (e.g., humanMERTK). After hybridoma cells that produce antibodies of the desiredspecificity, affinity, and/or activity are identified, the clones may besubcloned, grown, and separated from the culture medium by standardmethods (Goding J W (Ed), Monoclonal Antibodies: Principles andPractice, supra). The binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by methods known in the art,for example, immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay(ELISA).

In specific embodiments, disclosed herein are monoclonal antibodies thatare produced by a single cell (e.g., hybridoma or host cell producing arecombinant antibody), wherein the antibody immunospecifically binds toMERTK (e.g., human MERTK) as determined, e.g., by ELISA or otherantigen-binding or competitive binding assay known in the art or asdescribed in Section 6 herein. In certain embodiments, a monoclonalantibody is a monovalent antibody or multivalent (e.g., bivalent)antibody. In particular embodiments, a monoclonal antibody is amonospecific or multispecific antibody (e.g., bispecific antibody).

Antibody fragments which recognize MERTK (e.g., human MERTK) can begenerated by any technique known to those of skill in the art. Forexample, Fab and F(ab′)2 fragments described herein can be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments). A Fab fragment corresponds to one of the two identical armsof an antibody molecule and contains the complete light chain pairedwith the VH and C_(H)1 domains of the heavy chain. A F(ab′)2 fragmentcontains the two antigen-binding arms of an antibody molecule linked bydisulfide bonds in the hinge region.

Further, an anti-MERTK antibody or antigen-binding fragment thereofdescribed herein, can also be generated using various phage displaymethods known in the art. In phage display methods, functional antibodydomains are displayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. In particular, DNA sequencesencoding VH and VL domains are amplified from animal cDNA libraries(e.g., human or murine cDNA libraries of affected tissues). The DNAencoding the VH and VL domains are recombined together with a scFvlinker by PCR and cloned into a phagemid vector. The vector iselectroporated into E. coli cells and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13, and the VH and VL domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to a particular antigencan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Examples ofphage display methods that can be used to make the antibodies describedherein include those disclosed in Brinkman U et al., (1995) J ImmunolMethods 182: 41-50; Ames R S et al., (1995) J Immunol Methods 184:177-186; Kettleborough C A et al., (1994) Eur J Immunol 24: 952-958;Persic L et al., (1997) Gene 187: 9-18; Burton D R & Barbas C F (1994)Advan Immunol 57: 191-280; PCT Application No. PCT/GB91/001134;International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO 97/13844; andU.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727, 5,733,743 and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including humanized antibodies, chimericantibodies, or any other desired antigen-binding fragment, and expressedin any desired host, including mammalian cells, insect cells, plantcells, yeast, and bacteria, e.g., as described below. Techniques torecombinantly produce antibody fragments such as Fab, Fab′ and F(ab′)2fragments can also be employed using methods known in the art such asthose disclosed in PCT publication No. WO 92/22324; Mullinax R L et al.,(1992) BioTechniques 12(6): 864-9; Sawai H et al., (1995) Am J ReprodImmunol 34: 26-34; and Better M et al., (1988) Science 240: 1041-1043.

In one aspect, to generate whole antibodies, PCR primers including VH orVL nucleotide sequences, a restriction site, and a flanking sequence toprotect the restriction site can be used to amplify the VH or VLsequences from a template, e.g., scFv clones. Utilizing cloningtechniques known to those of skill in the art, the PCR amplified VHdomains can be cloned into vectors expressing a VH constant region, andthe PCR amplified VL domains can be cloned into vectors expressing a VLconstant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mouse orrat monoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L(1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J ImmunolMethods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,4,816,397, and 6,331,415. In a specific embodiment, a chimeric antibodycomprises a variable heavy chain region (VH) and a variable light chainregion (VL), wherein the VH and VL each comprise the amino acidsequences of VH and VL, respectively, set forth in Table 13.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Ahumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₂, IgG₃ and IgG₄. Humanized antibodies can be produced using avariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 andEP 519596; Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G Met al., (1994) Prot Engineering 7(6): 805-814; and Roguska M A et al.,(1994) PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,International Publication No. WO 93/17105; Tan P et al., (2002) JImmunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al.,(1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996) ProteinEng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23 Supp):5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22; SandhuJ S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) J MolBiol 235(3): 959-73. See also U.S. Application Publication No. US2005/0042664 A1 (Feb. 24, 2005), which is incorporated by referenceherein in its entirety.

In a specific embodiment, a humanized antibody or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),comprises the VH and VL of antibody z10 (see Table 11), the VH and VL ofantibody z11 (see Table 12), or the VH and VL of antibody z13 (see Table13). Methods for making multispecific (e.g., bispecific antibodies) havebeen described, see, for example, U.S. Pat. Nos. 7,951,917; 7,183,076;8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713.

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well known in the art. See RiechmannL & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al.,(2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) JBiotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and InternationalPublication Nos. WO 94/04678, WO 94/25591 and WO 01/44301.

Further, antibodies that immunospecifically bind to a MERTK (e.g., humanMERTK) antigen can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” an antigen using techniques well known to thoseskilled in the art. (See, e.g., Greenspan N S & Bona C A (1989) FASEB J7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438).

In particular embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof described herein binds to the same or an overlappingepitope of MERTK (e.g., human MERTK) as an anti-MERTK antibody orantigen-binding fragment thereof described herein. In particularembodiments, an antibody described herein, which competitively blocks(e.g., in a dose-dependent manner) any one of the antibodies describedherein, (e.g., z10, z11, or z13, or xAb) from binding to MERTK (e.g.,human MERTK), is a human anti-MERTK antibody or antigen-binding fragmentthereof. Human antibodies can be produced using any method known in theart. For example, transgenic mice which are incapable of expressingfunctional endogenous immunoglobulins, but which can express humanimmunoglobulin genes, can be used. In particular, the human heavy andlight chain immunoglobulin gene complexes can be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion can be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of an antigen (e.g., MERTK). Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see, e.g., Lonberg N& Huszar D (1995) Int Rev Immunol 13:65-93. For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g.,International Publication Nos. WO 98/24893, WO 96/34096 and WO 96/33735;and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318 and 5,939,598. Examples of mice capableof producing human antibodies include the Xenomouse™ (Abgenix, Inc.;U.S. Pat. Nos. 6,075,181 and 6,150,184), the HuAb-Mouse™ (Mederex,Inc./Gen Pharm; U.S. Pat. Nos. 5,545,806 and 5,569,825), the TransChromo Mouse™ (Kirin) and the KM Mouse™ (Medarex/Kirin).

Human antibodies which specifically bind to MERTK (e.g., human MERTK)can be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887,4,716,111, and 5,885,793; and International Publication Nos. WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741.

In some embodiments, human antibodies can be produced using mouse-humanhybridomas. For example, human peripheral blood lymphocytes transformedwith Epstein-Barr virus (EBV) can be fused with mouse myeloma cells toproduce mouse-human hybridomas secreting human monoclonal antibodies,and these mouse-human hybridomas can be screened to determine ones whichsecrete human monoclonal antibodies that immunospecifically bind to atarget antigen (e.g., human MERTK). Such methods are known and aredescribed in the art, see, e.g., Shinmoto H et al., (2004)Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31.

In specific embodiments, the methods of screening and selectingantibodies or antigen-binding fragments thereof described herein, whichspecifically bind to MERTK (e.g., human MERTK) are as described inExample 1, infra.

Once an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein, has been produced, it can be purified by any methodknown in the art for purification of an immunoglobulin molecule, forexample, by chromatography (e.g., ion exchange, affinity, particularlyby affinity for the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies described herein can be fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein is isolated or purified. Generally, anisolated antibody is one that is substantially free of other antibodieswith different antigenic specificities than the isolated antibody. Forexample, in a particular embodiment, a preparation of an antibodydescribed herein is substantially free of cellular material and/orchemical precursors. The language “substantially free of cellularmaterial” includes preparations of an antibody in which the antibody isseparated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, an antibody that issubstantially free of cellular material includes preparations ofantibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1%(by dry weight) of heterologous protein (also referred to herein as a“contaminating protein”) and/or variants of an antibody, for example,different post-translational modified forms of an antibody or otherdifferent versions of an antibody (e.g., antibody fragments). When theantibody is recombinantly produced, it is also generally substantiallyfree of culture medium, i.e., culture medium represents less than about20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the proteinpreparation. When the antibody is produced by chemical synthesis, it isgenerally substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. Accordingly, suchpreparations of the antibody have less than about 30%, 20%, 10%, or 5%(by dry weight) of chemical precursors or compounds other than theantibody of interest. In a specific embodiment, antibodies describedherein are isolated or purified. 5.3.2. Polynucleotides

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding an anti-MERTK antibody or anantigen-binding fragment thereof described herein that specificallybinds to a MERTK (e.g., human MERTK) antigen, and vectors, e.g., vectorscomprising such polynucleotides for their efficient expression in hostcells (e.g., E. coli and mammalian cells). In some embodiments, apolynucleotide is isolated or purified.

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of other material,e.g., cellular material, culture medium, other nucleic acid molecules,chemical precursors and/or other chemicals.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding an anti-MERTK antibody or antigen-bindingfragment thereof described herein, which specifically binds to a MERTK(e.g., human MERTK) polypeptide and comprises an amino acid sequence asdescribed herein, as well as antibodies which compete with suchantibodies for binding to a MERTK (e.g., human MERTK) polypeptide (e.g.,in a dose-dependent manner), or which binds to the same or anoverlapping epitope as that of such antibodies.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody described herein.

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding an anti-MERTK antibody or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK), wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain variable region that comprises an aminoacid sequence described herein (SEQ ID NO: 105, SEQ ID NO: 107, SEQ IDNO: 108 or SEQ ID NO: 109). In certain embodiments, a polynucleotidedescribed herein comprises a nucleotide sequence encoding an anti-MERTKantibody or antigen-binding fragment thereof described herein, whichspecifically binds to MERTK (e.g., human MERTK), wherein the anti-MERTKantibody or antigen-binding fragment thereof comprises a light chainvariable region that comprises an amino acid sequence described herein(SEQ ID NO: 106 or SEQ ID NO: 111). In certain embodiments, apolynucleotide described herein comprises a nucleotide sequence encodingan anti-MERTK antibody or antigen-binding fragment thereof describedherein, which specifically binds to MERTK (e.g., human MERTK), whereinthe antibody or antigen-binding fragment thereof comprises a heavy chainvariable region that comprises the amino acid sequence of SEQ ID NO:105, and a light chain variable region that comprises the amino acidsequence of SEQ ID NO: 106. In certain embodiments, a polynucleotidedescribed herein comprises a nucleotide sequence encoding an anti-MERTKantibody or an antigen-binding fragment thereof described herein, whichspecifically binds to MERTK (e.g., human MERTK), wherein the antibody orantigen-binding fragment thereof comprises a heavy chain variable regionthat comprises the amino acid sequence of SEQ ID NO: 107 and a lightchain variable region that comprises the amino acid sequence of SEQ IDNO: 106. In certain embodiments, a polynucleotide described hereincomprises a nucleotide sequence encoding an anti-MERTK antibody orantigen-binding fragment thereof described herein, which specificallybinds to MERTK (e.g., human MERTK), wherein the antibody orantigen-binding fragment thereof comprises a heavy chain variable regionthat comprises the amino acid sequence of SEQ ID NO: 108 and a lightchain variable region that comprises the amino acid sequence of SEQ IDNO: 106. In certain embodiments, a polynucleotide described hereincomprises a nucleotide sequence encoding an anti-MERTK antibody orantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK), wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain variable region that comprises the aminoacid of SEQ ID No: 110 and a light chain variable region that comprisesthe amino acid sequence of SEQ ID NO: 111. In certain embodiments, apolynucleotide described herein comprises a nucleotide sequence encodingan anti-MERTK antibody or antigen-binding fragment thereof, whichspecifically binds to MERTK (e.g., human MERTK), wherein the antibody orantigen-binding fragment thereof comprises a heavy chain variable regionthat comprises the amino acid of SEQ ID NO: 109 and a light chainvariable region that comprises the amino acid sequence of SEQ ID NO:106.

In certain embodiments, a polynucleotide described herein encodes a VH,wherein the polynucleotide comprises a nucleic acid sequence of SEQ IDNO: 119, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123. In certainembodiments, a polynucleotide described herein encodes a VL, wherein thepolynucleotide comprises a nucleic acid sequence of SEQ ID NO: 120 orSEQ ID NO: 124. In a specific embodiment, a polynucleotide describedherein encodes a VH and a VL, wherein the polynucleotide comprises anucleic acid sequence of SEQ ID NO: 119, and wherein the polynucleotidecomprises the nucleic acid sequence of SEQ ID NO: 120. In anotherspecific embodiment, a polynucleotide described herein encodes a VH anda VL, wherein the polynucleotide comprises a nucleic acid sequence ofSEQ ID NO: 121, and wherein the polynucleotide comprises the nucleicacid sequence of SEQ ID NO: 120. In another specific embodiment, apolynucleotide described herein encodes a VH and a VL, wherein thepolynucleotide comprises a nucleic acid sequence of SEQ ID NO: 122, andwherein the polynucleotide comprises the nucleic acid sequence of SEQ IDNO: 120. In another embodiment, a polynucleotide described hereinencodes a VH and a VL, wherein the polynucleotide comprises a nucleicacid SEQ ID NO: 123 and wherein the polynucleotide comprises the nucleicacid sequence of SEQ ID NO: 124.

In specific aspects, provided herein is a polynucleotide comprising anucleotide sequence encoding an antibody comprising a light chain and aheavy chain, e.g., a separate light chain and heavy chain. With respectto the light chain, in a specific embodiment, a polynucleotide providedherein comprises a nucleotide sequence encoding an antibody describedherein comprising a human kappa light chain or a human lambda lightchain.

In specific aspects, a polynucleotide provided herein comprises anucleotide sequence encoding a light chain of an antibody, wherein thenucleotide sequence comprises a variable light chain region of SEQ IDNO: 106, and a human light chain constant region nucleotide sequence ofthe human kappa or lambda light chain. In another aspect, apolynucleotide provided herein comprises a nucleotide sequence encodinga light chain of an antibody, wherein the light chain comprises avariable light chain region of SEQ ID NO: 111, and a human light chainconstant region of the human kappa or lambda light chain.

In a particular embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichspecifically binds to MERTK (e.g., human MERTK), wherein the antibodycomprises a light chain, and wherein the amino acid sequence of thevariable region of the light chain can comprise any amino acid sequenceof SEQ ID NO: 106 or SEQ ID NO: 111, and wherein the constant region ofthe light chain comprises the amino acid sequence of a human kappa lightchain constant region. In another particular embodiment, apolynucleotide provided herein comprises a nucleotide sequence encodingan antibody described herein, which specifically binds to MERTK (e.g.,human MERTK) and comprises a light chain, wherein the amino acidsequence of the variable region of the light chain can comprise anyamino acid sequence of SEQ ID NO: 106 or SEQ ID NO: 111, and wherein theconstant region of the light chain comprises the amino acid sequence ofa human lambda light chain constant region. In another aspect, apolynucleotide provided herein comprises a nucleotide sequence encodinga light chain of an antibody, wherein the light chain comprises theamino acid sequence of SEQ ID NO: 113 or 118. In another aspect, apolynucleotide provided herein comprises a nucleotide sequence encodinga light chain of an antibody which specifically binds to MERTK (e.g.,human MERTK), wherein the nucleotide sequence comprises SEQ ID NO: 126or 130.

In a particular embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichspecifically binds to MERTK (e.g., human MERTK), wherein the antibodycomprises a heavy chain, wherein the heavy chain comprises amino acidsequence of SEQ ID NO: 105, and a heavy constant region. In anotherparticular embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichspecifically binds to MERTK (e.g., human MERTK) and comprises a heavychain, wherein the heavy chain comprises amino acid sequence of SEQ IDNO: 107, and a heavy constant region. In another particular embodiment,a polynucleotide provided herein comprises a nucleotide sequenceencoding an antibody described herein, which specifically binds to MERTK(e.g., human MERTK) and comprises a heavy chain, wherein the heavy chaincomprises amino acid sequence of SEQ ID NO: 108, and a heavy constantregion. In another particular embodiment, a polynucleotide providedherein comprises a nucleotide sequence encoding an antibody describedherein, which specifically binds to MERTK (e.g., human MERTK) andcomprises a heavy chain, wherein the heavy chain comprises amino acidsequence of SEQ ID NO: 109, and a heavy constant region. In anotherparticular embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichspecifically binds to MERTK (e.g., human MERTK) and comprises a heavychain, wherein the heavy chain comprises amino acid sequence of SEQ IDNO: 110, and a heavy constant region.

In a particular embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichspecifically binds to MERTK (e.g., human MERTK), wherein the antibodycomprises a heavy chain, wherein the heavy chain comprises amino acidsequence of SEQ ID NO: 112. In a particular embodiment, a polynucleotideprovided herein comprises a nucleotide sequence encoding an antibodydescribed herein, which specifically binds to MERTK (e.g., human MERTK),wherein the antibody comprises a heavy chain, wherein the heavy chaincomprises amino acid sequence of SEQ ID NO: 114. In a particularembodiment, a polynucleotide provided herein comprises a nucleotidesequence encoding an antibody described herein, which specifically bindsto MERTK (e.g., human MERTK), wherein the antibody comprises a heavychain, wherein the heavy chain comprises amino acid sequence of SEQ IDNO: 115. In a particular embodiment, a polynucleotide provided hereincomprises a nucleotide sequence encoding an antibody described herein,which specifically binds to MERTK (e.g., human MERTK), wherein theantibody comprises a heavy chain, wherein the heavy chain comprisesamino acid sequence of SEQ ID NO: 116. In a particular embodiment, apolynucleotide provided herein comprises a nucleotide sequence encodingan antibody described herein, which specifically binds to MERTK (e.g.,human MERTK), wherein the antibody comprises a heavy chain, wherein theheavy chain comprises amino acid sequence of SEQ ID NO: 117.

In a specific aspect, polynucleotide provided herein comprises anucleotide sequence encoding a heavy chain of an antibody, whichspecifically binds to MERTK (e.g., human MERTK), wherein the nucleotidesequence comprises SEQ ID NO: 119 and a human heavy chain constantregion nucleotide sequence. In another aspect, a polynucleotide providedherein comprises a nucleotide sequence encoding a heavy chain of anantibody which specifically binds MERTK, wherein the nucleotide sequencecomprises SEQ ID NO: 125.

In a specific aspect, polynucleotide provided herein comprises anucleotide sequence encoding a heavy chain of an antibody, whichspecifically binds to MERTK (e.g., human MERTK), wherein the nucleotidesequence comprises SEQ ID NO: 121 and a human heavy chain constantregion nucleotide sequence. In another aspect, a polynucleotide providedherein comprises a nucleotide sequence encoding a heavy chain of anantibody which specifically binds MERTK, wherein the nucleotidecomprises SEQ ID NO: 127.

In a specific aspect, polynucleotide provided herein comprises anucleotide sequence encoding a heavy chain of an antibody, whichspecifically binds to MERTK (e.g., human MERTK), wherein the nucleotidesequence comprises SEQ ID NO: 122 and a human heavy chain constantregion nucleotide sequence. In another aspect, a polynucleotide providedherein comprises a nucleotide sequence encoding a heavy chain of anantibody which specifically binds MERTK (e.g., human MERTK), wherein thenucleotide sequence comprises SEQ ID NO: 128.

In a specific aspect, polynucleotide provided herein comprises anucleotide sequence encoding a heavy chain of an antibody, whichspecifically binds to MERTK (e.g. human MERTK), wherein the nucleotidesequence comprises SEQ ID NO: 123 and a human heavy chain constantregion nucleotide sequence. In another aspect, a polynucleotide providedherein comprises a nucleotide sequence encoding a heavy chain of anantibody which specifically binds MERTK (e.g. human MERTK), wherein thenucleotide comprises SEQ ID NO: 129.

In a specific aspect, a polynucleotide provided herein comprises anucleotide sequence encoding light chain of an antibody described hereinthat specifically binds to MERTK (e.g., human MERTK) and a nucleotidesequence encoding a heavy of chain of the antibody, wherein thenucleotide sequence encoding the heavy chain comprises SEQ ID NO: 119and a human heavy chain constant region nucleotide sequence, and whereinthe nucleotide sequence encoding the light chain comprises SEQ ID NO:120, and a human heavy chain constant region nucleotide sequence. Inanother specific aspects, a polynucleotide provided herein comprises anucleotide sequence encoding light chain of an antibody described hereinthat specifically binds to MERTK (e.g., human MERTK) and a nucleotidesequence encoding a heavy of chain of the antibody, wherein thenucleotide sequence encoding the light chain comprises SEQ ID NO: 120,and a human light chain constant region nucleotide sequence, and whereinthe nucleotide sequence encoding the heavy chain comprises SEQ ID NO:121 and a human heavy chain constant region nucleotide sequence. Inanother specific aspects, a polynucleotide provided herein comprises anucleotide sequence encoding light chain of an antibody described hereinthat specifically binds to MERTK (e.g., human MERTK) and a nucleotidesequence encoding a heavy of chain of the antibody, wherein thenucleotide sequence encoding the light chain comprises SEQ ID NO: 120and a human light chain constant region nucleotide sequence, and whereinthe nucleotide sequence encoding tge heavy chain comprises SEQ ID NO:122 and a human heavy chain constant region nucleotide sequence. Inanother specific aspects, a polynucleotide provided herein comprises anucleotide sequence encoding light chain of an antibody described hereinthat specifically binds to MERTK (e.g., human MERTK) and a nucleotidesequence encoding a heavy of chain of the antibody, wherein thenucleotide sequence encoding the light chain comprises SEQ ID NO: 124and a human light chain constant region nucleotide sequence, and whereinthe nucleotide sequence encoding the heavy chain comprises SEQ ID NO:123 and a human heavy chain constant region nucleotide sequence.

In a specific aspect, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody, which specifically binds toMERTK (e.g., human MERTK), wherein the antibody comprises: (1) avariable heavy chain region comprising the amino acid sequence of SEQ IDNO: 105 and a human heavy chain constant region; and (2) a variablelight chain region comprising the amino acid sequence of SEQ ID NO: 106,and a human light chain constant region. In another specific aspect, apolynucleotide provided herein comprises a nucleotide sequence encodingan antibody, which specifically binds to MERTK (e.g., human MERTK),wherein the antibody comprises: (1) a variable heavy chain regioncomprising the amino acid sequence of SEQ ID NO: 107 and a human heavychain constant region; and (2) a variable light chain region comprisingthe amino acid sequence of SEQ ID NO: 106, and a human light chainconstant region. In another specific aspect, a polynucleotide providedherein comprises a nucleotide sequence encoding an antibody, whichspecifically binds to MERTK (e.g., human MERTK), wherein the antibodycomprises: (1) a variable heavy chain region comprising the amino acidsequence of SEQ ID NO: 108 and a human heavy chain constant region; and(2) a variable light chain region comprising the amino acid sequence ofSEQ ID NO: 106, and a human light chain constant region. In anotherspecific aspect, a polynucleotide provided herein comprises a nucleotidesequence encoding an antibody, which specifically binds to MERTK (e.g.,human MERTK), wherein the antibody comprises: (1) a variable heavy chainregion comprising the amino acid sequence of SEQ ID NO: 110, and a humanheavy chain constant region; and (2) a variable light chain regioncomprising the amino acid sequence of SEQ ID NO: 111 and a human lightchain constant region.

In another specific aspect, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody, which specifically binds toMERTK (e.g., human MERTK), wherein the antibody comprises: (1) avariable heavy chain region comprising the amino acid sequence of SEQ IDNO: 109, and a human heavy chain constant region; and (2) a variablelight chain region comprising the amino acid sequence of SEQ ID NO: 106and a human light chain constant region.

In another specific embodiment, a polynucleotide provided hereincomprises nucleotide sequences encoding an anti-MERTK antibody, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK), wherein one nucleotide sequence encodes a heavychain of the antibody and the other nucleotide sequence encodes a lightchain of the antibody, and wherein the nucleotide sequence encoding theheavy chain comprises SEQ ID NO: 125 and the nucleotide sequenceencoding the light chain comprises SEQ ID NO: 126. In another specificembodiment, a polynucleotide provided herein comprises nucleotidesequences encoding an anti-MERTK antibody, or an antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK),wherein one nucleotide sequence encodes a heavy chain of the antibodyand the other nucleotide sequence encodes a light chain of the antibody,and wherein the nucleotide sequence encoding the heavy chain comprisesSEQ ID NO: 127 and the nucleotide sequence encoding the light chaincomprises SEQ ID NO: 126. In another specific embodiment, apolynucleotide provided herein comprises nucleotide sequences encodingan anti-MERTK antibody, or an antigen-binding fragment thereof, whichspecifically binds to MERTK (e.g., human MERTK), wherein one nucleotidesequence encodes a heavy chain of the antibody and the other nucleotidesequence encodes a light chain of the antibody, and wherein thenucleotide sequence encoding the heavy chain comprises SEQ ID NO: 128and the nucleotide sequence encoding the light chain comprises SEQ IDNO: 126. In another specific embodiment, a polynucleotide providedherein comprises nucleotide sequences encoding an anti-MERTK antibody,or an antigen-binding fragment thereof, which specifically binds toMERTK (e.g., human MERTK), wherein one nucleotide sequence encodes aheavy chain of the antibody and the other nucleotide sequence encodes alight chain of the antibody, and wherein the nucleotide sequenceencoding the heavy chain comprises SEQ ID NO: 129 and the nucleotidesequence encoding the light chain comprises SEQ ID NO: 130.

In another specific embodiment, a polynucleotide provided hereincomprises nucleotide sequences encoding an anti-MERTK antibody orantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK), wherein one nucleotide sequence encodes a heavychain of the antibody and the other encodes the light chain of theantibody, wherein the heavy chain comprises the amino acid sequence ofSEQ ID NO: 112 and the light chain comprises the amino acid sequence ofSEQ ID NO: 113. In another specific embodiment, a polynucleotideprovided herein comprises nucleotide sequences encoding an anti-MERTKantibody or antigen-binding fragment thereof, which specifically bindsto MERTK (e.g., human MERTK), wherein one nucleotide sequence encodes aheavy chain of the antibody and the other encodes the light chain of theantibody, wherein the heavy chain comprises the amino acid sequence ofSEQ ID NO: 114 and the light chain comprises the amino acid sequence ofSEQ ID NO: 113. In another specific embodiment, a polynucleotideprovided herein comprises nucleotide sequences encoding an anti-MERTKantibody or antigen-binding fragment thereof, which specifically bindsto MERTK (e.g., human MERTK), wherein one nucleotide sequence encodes aheavy chain of the antibody and the other encodes the light chain of theantibody, wherein the heavy chain comprises the amino acid sequence ofSEQ ID NO: 115 and the light chain comprises the amino acid sequence ofSEQ ID NO: 113. In another specific embodiment, a polynucleotideprovided herein comprises nucleotide sequences encoding an anti-MERTKantibody or antigen-binding fragment thereof, which specifically bindsto MERTK (e.g., human MERTK), wherein one nucleotide sequence encodes aheavy chain of the antibody and the other encodes the light chain of theantibody, wherein the heavy chain comprises the amino acid sequence ofSEQ ID NO: 116 and the light chain comprises the amino acid sequence ofSEQ ID NO: 113. In another specific embodiment, a polynucleotideprovided herein comprises nucleotide sequences encoding an anti-MERTKantibody or antigen-binding fragment thereof, which specifically bindsto MERTK (e.g., human MERTK), wherein one nucleotide sequence encodes aheavy chain of the antibody and the other encodes the light chain of theantibody, wherein the heavy chain comprises the amino acid sequence ofSEQ ID NO: 117 and the light chain comprises the amino acid sequence ofSEQ ID NO: 118.

In certain embodiments, a polynucleotide(s), nucleic acid(s) ornucleotide(s) includes deoxyribonucleic acids, ribonucleic acids,ribonucleotides, and polymeric forms thereof. In some embodiments, thepolynucleotides(s), nucleic acid(s) or nucleotide(s) is single or doublestranded. In a specific embodiment, a polynucleotide, nucleic acid, ornucleotide sequence is a cDNA sequence.

In some embodiments, a polynucleotide sequence described herein (e.g., anucleic acid sequence) encoding an anti-MERTK antibody or anantigen-binding fragment thereof is codon optimized using methodologyknown to one of skill in the art. In certain embodiments, an optimizedpolynucleotide sequence encoding an anti-MERTK antibody orantigen-binding fragment thereof described herein or an antigen-bindingfragment thereof (e.g., VH domain and/or VL domain) can hybridize to anantisense (e.g., complementary) polynucleotide of an unoptimizedpolynucleotide sequence encoding an anti-MERTK antibody described hereinor an antigen-binding fragment thereof (e.g., VH domain and/or VLdomain). In specific embodiments, an optimized nucleotide sequenceencoding an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein, hybridizes under high stringency conditions toantisense polynucleotide of an unoptimized polynucleotide sequenceencoding an anti-MERTK antibody described herein or an antigen-bindingfragment thereof. In a specific embodiment, an optimized nucleotidesequence encoding an anti-MERTK antibody or an antigen-binding fragmentthereof hybridizes under high stringency, intermediate or lowerstringency hybridization conditions to an antisense polynucleotide of anunoptimized nucleotide sequence encoding an anti-MERTK antibody or anantigen-binding fragment thereof described herein. Information regardinghybridization conditions has been described, see, e.g., U.S. PatentApplication Publication No. US 2005/0048549 (e.g., paragraphs 72-73),which is incorporated herein by reference.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding antibodies described herein, and modified versions ofthese antibodies can be determined using methods well known in the art,i.e., nucleotide codons known to encode particular amino acids areassembled in such a way to generate a nucleic acid sequence that encodesthe antibody. Such a polynucleotide encoding the antibody can beassembled from chemically synthesized oligonucleotides (e.g., asdescribed in Kutmeier G et al., (1994), BioTechniques 17: 242-6), which,briefly, involves the synthesis of overlapping oligonucleotidescontaining portions of the sequence encoding the antibody, annealing andligating of those oligonucleotides, and then amplification of theligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described hereincan be generated from nucleic acid from a suitable source (e.g., ahybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the antibody of interest. Such PCR amplification methods canbe used to obtain nucleic acids comprising the sequence encoding thelight chain and/or heavy chain of an antibody. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody. The amplified nucleic acids can be cloned intovectors for expression in host cells and for further cloning, forexample, to generate humanized antibodies.

If a clone containing a nucleic acid sequence encoding a particularantibody is not available, but the sequence of the antibody molecule isknown, a nucleic acid encoding the immunoglobulin can be chemicallysynthesized or obtained from a suitable source (e.g., an antibody cDNAlibrary or a cDNA library generated from, or nucleic acid, preferablypoly A+ RNA, isolated from, any tissue or cells expressing the antibody,such as hybridoma cells selected to express an antibody describedherein) by PCR amplification using synthetic primers hybridizable to the3′ and 5′ ends of the sequence or by cloning using an oligonucleotideprobe specific for the particular gene sequence to identify, e.g., acDNA clone from a cDNA library that encodes the antibody. Amplifiednucleic acids generated by PCR can then be cloned into replicablecloning vectors using any method well known in the art.

DNA encoding anti-MERTK antibodies described herein can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the anti-MERTK antibodies).Hybridoma cells can serve as a source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)),293F cells, HEK293 cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of MERTK agonisticantibodies in the recombinant host cells.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. In certain embodiments, the vectors for expressing the VH or VLdomains comprise a promoter, a secretion signal, a cloning site for thevariable domain, constant domains, and a selection marker. The VH and VLdomains can also be cloned into one vector expressing the necessaryconstant regions. The heavy chain conversion vectors and light chainconversion vectors are then co-transfected into cell lines to generatestable or transient cell lines that express full-length antibodies,e.g., IgG, using techniques known to those of skill in the art.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody described herein or anantigen-binding fragment thereof. In specific embodiments,polynucleotides described herein hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH (SEQ ID NO: 119, 121, 122 or 123) and/orVL (SEQ ID NO: 120 or 124) provided herein.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel F M et al., eds., (1989)Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3.

5.3.3. Cells and Vectors

In certain aspects, provided herein are vectors (e.g., expressionvectors) comprising polynucleotides comprising nucleotide sequencesencoding an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein for recombinant expression in host cells, preferably inmammalian cells. Expression vectors may be, e.g., plasmids or viralvectors (such as Newcastle disease virus, adenovirus, adeno-associatedvirus, vaccinia, etc.). Also provided herein are host cells comprisingsuch vectors for recombinantly expressing anti-MERTK antibodies orantigen-binding fragments thereof described herein.

Recombinant expression of an antibody described herein (e.g., afull-length antibody, heavy and/or light chain of an antibody, or asingle chain antibody described herein) that specifically binds to MERTK(e.g., human MERTK) involves construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule, heavy and/or light chainof an antibody, or an antigen-binding fragment thereof (e.g., heavyand/or light chain variable regions) described herein has been obtained,the vector for the production of the antibody molecule can be producedby recombinant DNA technology using techniques well known in the art.Thus, methods for preparing a protein by expressing a polynucleotidecontaining an antibody or antibody fragment (e.g., light chain or heavychain, or both) encoding nucleotide sequence are described herein.Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing antibody or antibody fragment(e.g., light chain or heavy chain, or both) coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Also providedare replicable vectors comprising a nucleotide sequence encoding anantibody molecule described herein, a heavy or light chain of anantibody, or a heavy or light chain variable domain of an anti-MERTKantibody or antigen-binding fragment thereof, operably linked to apromoter. Such vectors can, for example, include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,International Publication Nos. WO 86/05807 and WO 89/01036; and U.S.Pat. No. 5,122,464) and variable domains of the antibody can be clonedinto such a vector for expression of the entire heavy, the entire lightchain, or both the entire heavy and light chains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an anti-MERTK antibody describedherein or an antigen-binding fragment thereof. Thus, provided herein arehost cells containing a polynucleotide encoding an antibody describedherein or an antigen-binding fragment thereof, or a heavy or light chainthereof, or a fragment thereof, or a single chain antibody describedherein, operably linked to a promoter for expression of such sequencesin the host cell. As used herein, the term “host cell” can be any typeof cell, e.g., a primary cell, a cell in culture, or a cell from a cellline. In specific embodiments, the term “host cell” refers to a celltransfected with a nucleic acid molecule and the progeny or potentialprogeny of such a cell. Progeny of such a cell may not be identical tothe parent cell transfected with the nucleic acid molecule, e.g., due tomutations or environmental influences that may occur in succeedinggenerations or integration of the nucleic acid molecule into the hostcell genome.

A variety of host-expression vector systems can be utilized to expressantibody molecules described herein. Such host-expression systemsrepresent vehicles by which the coding sequences of interest can beproduced and subsequently purified, but also represent cells which can,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule described herein in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems (e.g., green algae such as Chlamydomonasreinhardtii) infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing antibody coding sequences; or mammalian cell systems(e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6,VERO, CRL7030, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, 293F, HepG2,SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 and BMT10 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). In a specific embodiment, cells for expressingantibodies described herein or an antigen-binding fragment thereof areCHO cells, for example CHO cells from the CHO GS System™ (Lonza). In aparticular embodiment, cells for expressing antibodies described hereinare human cells, e.g., human cell lines. In a specific embodiment, amammalian expression vector is pOptiVEC™ or pcDNA3.3. In a particularembodiment, bacterial cells such as Escherichia coli, or eukaryoticcells (e.g., mammalian cells), especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary (CHO) cells, in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking M K & Hofstetter H (1986) Gene 45: 101-5; and Cockett M I etal., (1990) Biotechnology 8(7): 662-7). In certain embodiments,antibodies described herein are produced by CHO cells or NS0 cells. In aspecific embodiment, the expression of nucleotide sequences encodingantibodies described herein which immunospecifically bind MERTK (e.g.,human MERTK) is regulated by a constitutive promoter, inducible promoteror tissue specific promoter.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983)EMBO J 2: 1791-1794), in which the antibody coding sequence can beligated individually into the vector in frame with the lac Z codingregion so that a fusion protein is produced; pIN vectors (Inouye S &Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster S M(1989) J Biol Chem 24: 5503-5509); and the like. For example, pGEXvectors can also be used to express foreign polypeptides as fusionproteins with glutathione 5-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to matrix glutathione agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV), for example, can be used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The antibody codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan J &Shenk T (1984) PNAS 81(12): 3655-9). Specific initiation signals canalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression can be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol.153: 516-544).

In addition, a host cell strain which modulates the expression of theinserted sequences, or modifies and processes the gene product in thespecific fashion desired can be chosen. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, 293F, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (amurine myeloma cell line that does not endogenously produce anyimmunoglobulin chains), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO,HsS78Bst, HEK-293T, HEK293, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40,YB/20, BMT10 and HsS78Bst cells. In certain embodiments, anti-MERTKantibodies described herein are produced in mammalian cells, such as CHOcells.

In a specific embodiment, the antibodies described herein orantigen-binding fragments thereof have reduced fucose content or nofucose content. Such antibodies can be produced using techniques knownone skilled in the art. For example, the antibodies can be expressed incells deficient or lacking the ability of to fucosylate. In a specificexample, cell lines with a knockout of both alleles ofα1,6-fucosyltransferase can be used to produce antibodies orantigen-binding fragments thereof with reduced fucose content. ThePotelligent® system (Lonza) is an example of such a system that can beused to produce antibodies or antigen-binding fragments thereof withreduced fucose content.

For long-term, high-yield production of recombinant proteins, stableexpression cells can be generated. For example, cell lines which stablyexpress an anti-MERTK antibody described herein or an antigen-bindingfragment thereof or a chimeric anti-MERTK antibody or antigen-bindingfragment thereof described herein, can be engineered. In specificembodiments, a cell provided herein stably expresses a light chain/lightchain variable domain and a heavy chain/heavy chain variable domainwhich associate to form an antibody described herein or anantigen-binding fragment thereof.

In certain aspects, rather than using expression vectors which containviral origins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA/polynucleotide, engineered cells can be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express a humanized anti-MERTKantibody described herein or an antigen-binding fragment thereof or achimeric anti-MERTK antibody or antigen-binding fragment thereofdescribed herein,. Such engineered cell lines can be particularly usefulin screening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems can be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (SzybalskaE H & Szybalski W (1962) PNAS 48(12): 2026-2034) and adeninephosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23)genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (WiglerM et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance tothe aminoglycoside G-418 (Wu G Y & Wu C_(H) (1991) Biotherapy 3: 87-95;Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan R C(1993) Science 260: 926-932; and Morgan R A & Anderson W F (1993) AnnRev Biochem 62: 191-217; Nabel G J & Felgner P L (1993) TrendsBiotechnol 11(5): 211-5); and hygro, which confers resistance tohygromycin (Santerre R F et al., (1984) Gene 30(1-3): 147-56). Methodscommonly known in the art of recombinant DNA technology can be routinelyapplied to select the desired recombinant clone and such methods aredescribed, for example, in Ausubel F M et al., (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler M, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, N Y(1990); and in Chapters 12 and 13, Dracopoli N C et al., (eds.), CurrentProtocols in Human Genetics, John Wiley & Sons, N Y (1994);Colbere-Garapin F et al., (1981) J Mol Biol 150: 1-14, which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington C R & Hentschel C C G, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse G F et al., (1983) Mol Cell Biol3: 257-66).

The host cell can be co-transfected with two or more expression vectorsdescribed herein, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides. Thehost cells can be co-transfected with different amounts of the two ormore expression vectors. For example, host cells can be transfected withany one of the following ratios of a first expression vector and asecond expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

In a specific aspect, a host cell provided herein comprises a vector,wherein the vector comprises a nucleotide sequence encoding a variablelight chain region (VL) of an antibody described herein thatspecifically binds to MERTK (e.g., human MERTK) and a nucleotidesequence encoding a variable heavy chain region (VH) of the antibody,wherein the VL comprises the amino acid sequence of SEQ ID NO: 106, andwherein the VH comprises the amino acid sequence of SEQ ID NO: 105. Inanother specific aspect, a host cell provided herein comprises a vector,wherein the vector comprises a nucleotide sequence encoding light chainof an antibody described herein that specifically binds to MERTK (e.g.,human MERTK) and a nucleotide sequence encoding a heavy chain of theantibody, wherein the light chain comprises a variable light chainregion comprising the amino acid of SEQ ID NO: 106, and a human lightchain constant region, and wherein the heavy chain comprises a variableheavy chain region comprising the amino acid sequence of SEQ ID NO: 105,and a human heavy chain constant region.

In a specific aspect, a host cell provided herein comprises a vector,wherein the vector comprises a nucleotide sequence encoding a variablelight chain region (VL) of an antibody described herein thatspecifically binds to MERTK (e.g., human MERTK) and a nucleotidesequence encoding a variable heavy chain region (VH) of the antibody,wherein the VL comprises the amino acid sequence of SEQ ID NO: 106, andwherein the VH comprises the amino acid sequence of SEQ ID NO: 109 Inanother specific aspect, a host cell provided herein comprises a vector,wherein the vector comprises a nucleotide sequence encoding light chainof an antibody described herein that specifically binds to MERTK (e.g.,human MERTK) and a nucleotide sequence encoding a heavy chain of theantibody, wherein the light chain comprises a variable light chainregion comprising the amino acid of SEQ ID NO: 106, and a human lightchain constant region, and wherein the heavy chain comprises a variableheavy chain region comprising the amino acid sequence of SEQ ID NO: 109,and a human heavy chain constant region.

In a specific aspect, a host cell provided herein comprises a vector,wherein the vector comprises a nucleotide sequence encoding VL of anantibody described herein that specifically binds to MERTK (e.g., humanMERTK) and a nucleotide sequence encoding a VH of the antibody, whereinthe VL comprises the amino acid sequence of SEQ ID NO: 106, and whereinthe VH comprises the amino acid sequence of SEQ ID NO: 107. In anotherspecific aspect, a host cell provided herein comprises a vector, whereinthe vector comprises a nucleotide sequence encoding the light chain ofan antibody described herein that specifically binds to MERTK (e.g.,human MERTK) and a nucleotide sequence encoding a heavy chain of theantibody, wherein the light chain comprises a VL comprising the aminoacid sequence of SEQ ID NO: 106 and a human light constant region, andwherein the heavy chain comprises a VH comprising the amino acidsequence of SEQ ID NO: 107 and a human heavy constant region.

In a specific aspect, a host cell provided herein comprises a vector,wherein the vector comprises a nucleotide sequence encoding a VL of anantibody described herein that specifically binds to MERTK (e.g., humanMERTK) and a nucleotide sequence encoding a VH of the antibody, whereinthe VL comprises the amino acid sequence of SEQ ID NO: 106, and whereinthe VH comprises the amino acid sequence of SEQ ID NO: 108. In anotherspecific aspect, a host cell provided herein comprises a vector, whereinthe vector comprises a nucleotide sequence encoding light chain of anantibody described herein that specifically binds to MERTK (e.g., humanMERTK) and a nucleotide sequence encoding a heavy chain of the antibody,wherein the light chain comprises a variable light chain region of SEQID NO: 108, and a human light chain constant region, and wherein theheavy chain comprises a variable heavy chain region nucleotide sequenceof SEQ ID NO: 105, and a human heavy chain constant region.

In a specific aspect, a host cell provided herein comprises a vector,wherein the vector comprises a nucleotide sequence encoding a VL of anantibody described herein that specifically binds to MERTK (e.g., humanMERTK) and a nucleotide sequence encoding a VH of the antibody, whereinthe VL comprises the amino acid sequence of SEQ ID NO: 111, and whereinthe VH comprises the amino acid sequence of SEQ ID NO: 110.

In another specific aspect, a host cell provided herein comprises avector, wherein the vector comprises a nucleotide sequence encodinglight chain of an antibody described herein that specifically binds toMERTK (e.g., human MERTK) and a nucleotide sequence encoding a heavychain of the antibody, wherein the nucleotide sequence encoding thelight chain comprises SEQ ID NO: 120, and a human light chain constantregion nucleotide sequence, and wherein the nucleotide sequence encodingthe heavy chain comprises of SEQ ID NO: 119, and a human heavy chainconstant region nucleotide sequence.

In another specific aspect, a host cell provided herein comprises avector, wherein the vector comprises a nucleotide sequence encodinglight chain of an antibody described herein that specifically binds toMERTK (e.g., human MERTK) and a nucleotide sequence encoding a heavychain of the antibody, wherein the nucleotide sequence encoding thelight chain comprises SEQ ID NO: 120, and a human light chain constantregion nucleotide sequence, and wherein the nucleotide sequence encodingthe heavy chain comprises of SEQ ID NO: 121, and a human heavy chainconstant region nucleotide sequence. In another specific aspect, a hostcell provided herein comprises a vector, wherein the vector comprises anucleotide sequence encoding light chain of an antibody described hereinthat specifically binds to MERTK (e.g., human MERTK) and a nucleotidesequence encoding a heavy chain of the antibody, wherein the nucleotidesequence encoding the light chain comprises SEQ ID NO: 120, and a humanlight chain constant region nucleotide sequence, and wherein thenucleotide sequence encoding the heavy chain comprises of SEQ ID NO:122, and a human heavy chain constant region nucleotide sequence. Inanother specific aspect, a host cell provided herein comprises a vector,wherein the vector comprises a nucleotide sequence encoding light chainof an antibody described herein that specifically binds to MERTK (e.g.,human MERTK) and a nucleotide sequence encoding a heavy chain of theantibody, wherein the nucleotide sequence encoding the light chaincomprises SEQ ID NO: 124, and a human light chain constant regionnucleotide sequence, and wherein the nucleotide sequence encoding theheavy chain comprises of SEQ ID NO: 123, and a human heavy chainconstant region nucleotide sequence.

In a specific aspect, a host cell provided herein comprises a firstvector and a second vector, wherein the first vector comprises anucleotide sequence encoding a variable heavy chain region of anantibody described herein, which specifically binds to MERTK (e.g.,human MERTK), wherein the second vector comprises a variable light chainregion of the antibody, wherein the variable heavy chain regioncomprises the amino acid sequence of SEQ ID NO: 105, and wherein thevariable light chain region comprises the amino acid sequence of SEQ IDNO: 106. In another specific aspect, a host cell provided hereincomprises a first vector and a second vector, wherein the first vectorcomprises a nucleotide sequence encoding a variable heavy chain regionof an antibody described herein, which specifically binds to MERTK(e.g., human MERTK), wherein the second vector comprises a variablelight chain region of the antibody, wherein the variable heavy chainregion comprises the amino acid sequence of SEQ ID NO: 107, and whereinthe variable light chain region comprises the amino acid sequence of SEQID NO: 106. In another specific aspect, a host cell provided hereincomprises a first vector and a second vector, wherein the first vectorcomprises a nucleotide sequence encoding a variable heavy chain regionof an antibody described herein, which specifically binds to MERTK(e.g., human MERTK), wherein the second vector comprises a variablelight chain region of the antibody, wherein the variable heavy chainregion comprises the amino acid sequence of SEQ ID NO: 108, and whereinthe variable light chain region comprises the amino acid sequence of SEQID NO: 106. In another specific aspect, a host cell provided hereincomprises a first vector and a second vector, wherein the first vectorcomprises a nucleotide sequence encoding a variable heavy chain regionof an antibody described herein, which specifically binds to MERTK(e.g., human MERTK), wherein the second vector comprises a variablelight chain region of the antibody, wherein the variable heavy chainregion comprises the amino acid sequence of SEQ ID NO: 109, and whereinthe variable light chain region comprises the amino acid sequence of SEQID NO: 106. In another specific aspect, a host cell provided hereincomprises a first vector and a second vector, wherein the first vectorcomprises a nucleotide sequence encoding a variable heavy chain regionof an antibody described herein, which specifically binds to MERTK(e.g., human MERTK), wherein the second vector comprises a variablelight chain region of the antibody, wherein the variable heavy chainregion comprises the amino acid sequence of SEQ ID NO: 110 and whereinthe variable light chain region comprises the amino acid sequence of SEQID NO: 111. In some embodiments, the first vector comprises a heavychain constant region and/or the second vector comprises a light chainconstant region.

Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In such anexpression vector, the transcription of both genes can be driven by acommon promoter, whereas the translation of the mRNA from the first genecan be by a cap-dependent scanning mechanism and the translation of themRNA from the second gene can be by a cap-independent mechanism, e.g.,by an IRES.

In a specific aspect, a host cell provided herein comprises a vector,wherein the vector comprises a nucleotide sequence encoding a variableheavy chain region of an antibody described herein, which specificallybinds to MERTK (e.g., human MERTK), and a nucleotide encoding a variablelight chain region of the antibody, wherein the variable heavy chainregion comprises the amino acid sequence of SEQ ID NO: 105, and whereinthe variable light chain region comprises the amino acid sequence of SEQID NO: 106. In another specific aspect, a host cell provided hereincomprises a vector, wherein the vector comprises a nucleotide sequenceencoding a variable heavy chain region of an antibody described herein,which specifically binds to MERTK (e.g., human MERTK), and a nucleotideencoding a variable light chain region of the antibody, wherein thevariable heavy chain region comprises the amino acid sequence of SEQ IDNO: 107, and wherein the variable light chain region comprises the aminoacid sequence of SEQ ID NO: 106. In another specific aspect, a host cellprovided herein comprises a vector, wherein the vector comprises anucleotide sequence encoding a variable heavy chain region of anantibody described herein, which specifically binds to MERTK (e.g.,human MERTK), and a nucleotide encoding a variable light chain region ofthe antibody, wherein the variable heavy chain region comprises theamino acid sequence of SEQ ID NO: 108, and wherein the variable lightchain region comprises the amino acid sequence of SEQ ID NO: 106. Inanother specific aspect, a host cell provided herein comprises a vector,wherein the vector comprises a nucleotide sequence encoding a variableheavy chain region of an antibody described herein, which specificallybinds to MERTK (e.g., human MERTK), and a nucleotide encoding a variablelight chain region of the antibody, wherein the variable heavy chainregion comprises the amino acid sequence of SEQ ID NO: 109, and whereinthe variable light chain region comprises the amino acid sequence of SEQID NO: 106. In another specific aspect, a host cell provided hereincomprises a vector, wherein the vector comprises a nucleotide sequenceencoding a variable heavy chain region of an antibody described herein,which specifically binds to MERTK (e.g., human MERTK), and a nucleotideencoding a variable light chain region of the antibody, wherein thevariable heavy chain region comprises the amino acid sequence of SEQ IDNO: 110, and wherein the variable light chain region comprises the aminoacid sequence of SEQ ID NO: 111. In some embodiments, the vectorcomprises a heavy chain constant region and/or a light chain constantregion.

In a specific embodiment, a host cell (e.g., an ex vivo host cell)described herein is cultured under conditions to produce the antibody orantigen-binding fragment thereof encoded by the polynucleotide sequencecontained in the host cell using a technique known in the art. Incertain embodiments, the antibody or antigen-binding fragment thereof isisolated or purified from the host cell using a technique known in theart.

5.4. Production of the Antibody-Drug Conjugates

In another aspect, provided herein are methods of producing anantibody-drug conjugate described herein wherein a linker is notpresent, the method comprising: (a) conjugating the cytotoxic agentdirectly to the antibody moiety to produce the antibody-drug conjugate;and (b) purifying the antibody-drug conjugate. Examples of methods ofproducing antibody-drug conjugates are described in section 6 (e.g., inExample 4).

In another aspect, provided herein are methods of producing anantibody-drug conjugate described herein wherein the antibody-drugconjugate comprises a linker, the method comprising the following stepsin the order stated: (a) conjugating the linker directly to the antibodymoiety to produce a linker-antibody moiety; (b) conjugating the linkerof the linker-antibody moiety directly to the cytotoxic agent to producethe antibody-drug conjugate; and (c) purifying the antibody-drugconjugate.

In another aspect, provided herein are methods of producing anantibody-drug conjugate described herein wherein the antibody-drugconjugate comprises a linker, the method comprising the following stepsin the order stated: (a) conjugating the linker directly to thecytotoxic agent to produce a linker-cytotoxic agent moiety; (b)conjugating the linker of the linker-cytotoxic agent moiety directly tothe antibody moiety to produce the antibody-drug conjugate; and (c)purifying the antibody-drug conjugate.

The conjugating and purifying steps can be performed by methods known inthe art used for producing antibody-drug conjugates, such as the methodsdescribed in Beck A et al., (2017) Nat Rev Drug Discov 16: 315-337;Peters C and Brown S, (2015) Biosci Rep 35: art:e00225; McCombs J R andOwen S C (2015) The AAPS Journal 17: 339-351; Jackson D Y (2016) OrgProcess Res Dev 20: 852-866; or Olivier K J and Hurvitz S A ed., (2016)Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical,Wiley. In some embodiments, the drug moiety is conjugated to one chainof the antibody moiety (for example, when the antibody moiety is a scFv,or when the antibody moiety is a multi-chain antibody, such as animmunoglobulin (which is a tetramer), or antigen-binding fragmentthereof). In other embodiments, the drug moiety is conjugated to two ormore chains of the antibody moiety (when the antibody moiety is amulti-chain antibody, such as an immunoglobulin, or antigen-bindingfragment thereof). In a specific embodiment, the drug moiety isconjugated to two identical chains of an immunoglobulin, e.g., the heavychains or the light chains. In other embodiments, the drug moiety isconjugated to all chains of the antibody moiety (when the antibodymoiety is a multi-chain antibody, such as an immunoglobulin orantigen-binding fragment thereof). In a specific embodiment, theantibody-drug conjugate described herein is purified by chromatograph.In another specific embodiment, the antibody-drug conjugate describedherein is purified by centrifugation. By way of example but notlimitation, the conjugation chemistry that can be used for generatingthe antibody-drug conjugate can be thiol plus maleimide, thiol plusself-hydrolyzing maleimide, thiol plus phenyloxadiazole sulfone, oximeligation, alkoxyamine-to-keto-group reaction, strain-promotedazide-alkyne cycloaddition (SPAAC), copper-free click chemistry,cysteine oxidized to formylglycine, hydrazino-iso-Pictet-Spengler (HIPS)ligation, ligation of γ-carboxyamide group from glutamine residues plusprimary amines, ligation LPETG plus primary amine of polyglycine motif,maleimide plus 6-thiofucose, fucose-specific conjugation of hydrazide,periodate oxidation (aldehyde) plus amino-oxy-payload, strain-promotedalkyn-azide cycloaddition, C2-keto-gal oximation, site selectivealdehyde oxidation plus oxime ligation, oxime ligation NH2 plusindole-based 5-difluoro-2,4-dinitrobenzene derivatives, thiol plusbis-sulfone, thiol plus dibromomaleimide, thiol plus maleimide followedby pH 9.2 treatment (45° C., 48 hours), or thiol plus arylpropionitrile(see Beck A et al., (2017) Nat Rev Drug Discov 16: 315-337).

The anti-MERTK antibody or an antigen-binding fragment thereof describedherein can be generated as described in 5.3 infra. In specificembodiments, the anti-MERTK antibody or an antigen-binding fragmentthereof described herein is engineered or modified by a method known inthe art to facilitate conjugation with the drug moiety(ies), inparticular to facilitate site-specific conjugation with the drugmoiety(ies), for example, by a method described in Beck A et al., (2017)Nat Rev Drug Discov 16: 315-337; Peters C and Brown S, (2015) Biosci Rep35: art:e00225; McCombs J R and Owen S C (2015) The AAPS Journal 17:339-351; Jackson D Y (2016) Org Process Res Dev 20: 852-866; or OlivierK J and Hurvitz S A ed., (2016) Antibody-Drug Conjugates: Fundamentals,Drug Development, and Clinical, Wiley. Non-limiting exemplary methodsthat can be used (see Beck A et al., (2017) Nat Rev Drug Discov 16:315-337) to engineer or modify an anti-MERTK antibody or anantigen-binding fragment thereof to facilitate conjugation with the drugmoiety(ies) include adding one or more additional cysteines orselenocysteines, unnatural amino acid engineering, adding one or moreamino acid tags recognizable by certain enzymes that can assist with theconjugation, glycan remodeling, adding an amino-terminal serine, andnative cysteine bridging.

The linker and the cytotoxic agent can be generated by any method knownin the art for producing the linker and the cytotoxic agent,respectively.

5.5. Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising (a) ananti-MERTK antibody (e.g., a humanized antibody) or an antigen-bindingfragment thereof described herein and (b) a pharmaceutically acceptablecarrier. In a specific embodiment, the antibody or antigen-bindingfragment thereof is purified. In a specific embodiment, the antibody orantigen-binding fragment thereof is present in the pharmaceuticalcomposition in a therapeutically effective amount. In a specificembodiment, the pharmaceutical composition comprises an anti-MERTKantibody or an antigen-binding fragment thereof comprising a VHcomprising the amino acid sequence of SEQ ID NO: 105 and a VL comprisingthe amino acid sequence of SEQ ID NO: 106 (e.g., antibody z10). Inanother specific embodiment, the pharmaceutical composition comprises ananti-MERTK antibody or an antigen-binding fragment thereof comprising aVH comprising the amino acid sequence of SEQ ID NO: 107 and a VLcomprising the amino acid sequence of SEQ ID NO: 106 (e.g. antibodyz11). In another specific embodiment, the pharmaceutical compositioncomprises an anti-MERTK antibody or an antigen-binding fragment thereofcomprising a VH comprising the amino acid sequence of SEQ ID NO: 108 anda VL comprising the amino acid sequence of SEQ ID NO: 106 (e.g.,antibody z13). In another specific embodiment, the pharmaceuticalcomposition comprises an anti-MERTK antbody or antigen-binding fragmentthereof comprising the amino acid sequence of SEQ ID NO: 109 and a VLcomprising the amino acid sequence of SEQ ID NO: 106.

Also provided herein are pharmaceutical compositions comprising (a) achimeric anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein and (b) a pharmaceutically acceptable carrier. In aspecific embodiment, the antibody or antigen-binding fragment thereof ispurified. In a specific embodiment, the antibody or antigen-bindingfragment thereof is present in the pharmaceutical composition in atherapeutically effective amount. In a specific embodiment, thepharmaceutical composition comprises a chimeric antibody or anantigen-binding fragment thereof comprising a VH comprising the aminoacid sequence of SEQ ID NO: 110 and a VL comprising the amino acidsequence of SEQ ID NO: 111 (e.g., antibody xAb). Also provided hereinare pharmaceutical compositions comprising a polynucleotide or vector(s)described herein and a pharmaceutically acceptable carrier.

Also provided herein are pharmaceutical compositions comprising (a) anantibody-drug conjugate described herein and (b) a pharmaceuticallyacceptable carrier. In a specific embodiment, the antibody-drugconjugate is present in the pharmaceutical composition in atherapeutically effective amount.

Also provided herein are pharmaceutical compositions comprising (a) abispecific antibody that binds MERTK (e.g., human MERTK) and anotherantigen of interest (e.g., as described herein) and (b) apharmaceutically acceptable carrier. In a specific embodiment, thebispecific antibody is purified. In a specific embodiment, thebispecific antibody is present in the pharmaceutical composition in atherapeutically effective amount.

In a specific embodiment, a population of antibody-drug conjugatescontained in the pharmaceutical composition have a drug-to-antibodyratio (DAR) between 1 to 20. DAR is the average number of cytotoxicagents conjugated to the antibodies. In another specific embodiment, apopulation of antibody-drug conjugates contained in the pharmaceuticalcomposition have a DAR between 1 to 15. In another specific embodiment,a population of antibody-drug conjugates contained in the pharmaceuticalcomposition have a DAR between 1 to 12. In another specific embodiment,a population of antibody-drug conjugates contained in the pharmaceuticalcomposition have a DAR between 1 to 8. In another specific embodiment, apopulation of antibody-drug conjugates contained in the pharmaceuticalcomposition have a DAR between 3 to 5. In another specific embodiment, apopulation of antibody-drug conjugates contained in the pharmaceuticalcomposition have a DAR between 3.5 to 4.

Acceptable carriers, which can be excipients or stabilizers, arenontoxic to recipients at the dosages and concentrations employed, andinclude but are not limited to buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

In a specific embodiment, pharmaceutical compositions comprise ananti-MERTK antibody or an antigen-binding fragment thereof describedherein, and optionally one or more additional prophylactic ortherapeutic agents, in a pharmaceutically acceptable carrier. In aspecific embodiment, pharmaceutical compositions comprise an effectiveamount of an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein, and optionally one or more additional prophylactic ortherapeutic agents, in a pharmaceutically acceptable carrier. In aspecific embodiment, the pharmaceutical compositions and/or antibodiesor antigen-binding fragments thereof described herein, can be combinedwith a therapeutically effective amount of any of the additionaltherapeutic agents described herein (See Section 5.4.2, infra).

In a specific embodiment, pharmaceutical compositions comprise anantibody-drug conjugate described herein, and optionally one or moreadditional prophylactic or therapeutic agents, in a pharmaceuticallyacceptable carrier. In a specific embodiment, pharmaceuticalcompositions comprise an effective amount of an antibody-drug conjugatedescribed herein, and optionally one or more additional prophylactic ortherapeutic agents, in a pharmaceutically acceptable carrier. In aspecific embodiment, the pharmaceutical compositions and/orantibody-drug conjugate described herein, can be combined with atherapeutically effective amount of any of the additional therapeuticagents described herein (See Section 5.4.2, infra).

In some embodiments, the antibody-drug conjugate is the only activeingredient included in the pharmaceutical composition.

In some embodiments, an anti-MERTK antibody is the only activeingredient included in the pharmaceutical composition. In a specificembodiment, a humanized antibody described herein or an antigen-bindingfragment thereof is the only active ingredient included in thepharmaceutical composition. In another embodiment, a polynucleotide(s)or a vector(s) encoding an antibody or an antigen-binding fragmentthereof is the only active ingredient in the pharmaceutical composition.

Pharmaceutical compositions described herein can be used to treatcancer. Pharmaceutical compositions may be formulated for any route ofadministration (e.g., parenteral, topical, intratumoral, etc.).

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

A pharmaceutical composition may be formulated for any route ofadministration to a subject. Specific examples of routes ofadministration include intranasal, oral, pulmonary, transdermal,intradermal, and parenteral. Parenteral administration, characterized byeither subcutaneous, intramuscular or intravenous injection, is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of an antibody includesterile solutions ready for injection, sterile dry soluble products,such as lyophilized powders, ready to be combined with a solvent justprior to use, including hypodermic tablets, sterile suspensions readyfor injection, sterile dry insoluble products ready to be combined witha vehicle just prior to use and sterile emulsions. The solutions may beeither aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Topical mixtures comprising an antibody are prepared as described forthe local and systemic administration. The resulting mixture can be asolution, suspension, emulsions or the like and can be formulated ascreams, gels, ointments, emulsions, solutions, elixirs, lotions,suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,suppositories, bandages, dermal patches or any other formulationssuitable for topical administration.

An anti-MERTK antibody or an antigen-binding fragment thereof, or anantibody-drug conjugate described herein can be formulated as an aerosolfor topical application, such as by inhalation (see, e.g., U.S. Pat.Nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosols fordelivery of a steroid useful for treatment of inflammatory diseases,particularly asthma). These formulations for administration to therespiratory tract can be in the form of an aerosol or solution for anebulizer, or as a microfine powder for insufflations, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will, in one embodiment, have diameters ofless than 50 microns, in one embodiment less than 10 microns.

An anti-MERTK antibody or an antigen-binding fragment thereof, or anantibody-drug-conjugate described herein can be formulated for local ortopical application, such as for topical application to the skin andmucous membranes, such as in the eye, in the form of gels, creams, andlotions and for application to the eye or for intracisternal orintraspinal application. Topical administration is contemplated fortransdermal delivery and also for administration to the eyes or mucosa,or for inhalation therapies. Nasal solutions of the antibody alone or incombination with other pharmaceutically acceptable excipients can alsobe administered.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art, and can be used toadminister an antibody. For example, such patches are disclosed in U.S.Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975,6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

In certain embodiments, a pharmaceutical composition comprising ananti-MERTK antibody or an antigen-binding fragment thereof, or anantibody-drug conjugate described herein is a lyophilized powder, whichcan be reconstituted for administration as solutions, emulsions andother mixtures. It may also be reconstituted and formulated as solids orgels. The lyophilized powder is prepared by dissolving an anti-MERTKantibody or an antigen-binding fragment thereof, or an antibody-drugconjugate described herein, or a pharmaceutically acceptable derivativethereof, in a suitable solvent. In some embodiments, the lyophilizedpowder is sterile. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbitol, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

In some embodiments, pharmaceutical compositions comprising ananti-MERTK antibody or an antigen-binding fragment thereof describedherein, or an antibody-drug conjugate described herein, are supplied inliquid form without the need to reconstitute.

The antibodies or antigen-binding fragments thereof described herein, orantibody-drug conjugates described herein and other compositionsprovided herein can also be formulated to be targeted to a particulartissue, receptor, or other area of the body of the subject to betreated. Many such targeting methods are well known to those of skill inthe art. All such targeting methods are contemplated herein for use inthe instant compositions. For non-limiting examples of targetingmethods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,5,840,674, 5,759,542 and 5,709,874. In a specific embodiment, ananti-MERTK antibody or an antigen-binding fragment thereof describedherein, or an antibody-drug conjugate described herein is targeted to atumor.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

5.6 Uses and Methods

5.6.1 Therapeutic Uses and Methods

5.6.1.1 Cancer

In one aspect, presented herein are methods for treating cancer in asubject, comprising administering to a subject in need thereof ananti-MERTK antibody or antigen-binding fragment thereof describedherein, or a pharmaceutical composition comprising an anti-MERTKantibody or an antigen-binding fragment thereof described herein. In aspecific embodiment, the method for treating cancer comprisesadministering to a subject in need thereof an anti-MERTK antibodycomprising a VH comprising an amino acid sequence of SEQ ID NO: 105 anda VL comprising an amino acid sequence of SEQ ID NO: 106 (e.g., antibodyz10). In another specific embodiment, the method for treating cancercomprises administering to a subject in need thereof an anti-MERTKantibody comprising a VH comprising an amino acid sequence of SEQ ID NO:107 and a VL comprising an amino acid sequence of SEQ ID NO: 106 (e.g.,antibody z11). In a specific embodiment, the method for treating cancercomprises administering to a subject in need thereof an anti-MERTKantibody comprising a VH comprising an amino acid sequence of SEQ ID NO:108 and a VL comprising an amino acid sequence of SEQ ID NO: 106 (e.g.,antibody z13). In a specific embodiment, the method for treating cancercomprises administering to a subject in need thereof an anti-MERTKantibody comprising a VH comprising an amino acid sequence of SEQ ID NO:109 and a VL comprising an amino acid sequence of SEQ ID NO: 106 (e.g.,antibody z13).

In another aspect, presented herein are methods for treating cancer in asubject, comprising administering to a subject in need thereof achimeric anti-MERTK antibody or antigen-binding fragment thereofdescribed herein, or a pharmaceutical composition comprising a chimericanti-MERTK antibody or an antigen-binding fragment thereof describedherein. In a specific embodiment, the method for treating cancercomprises administering to a subject in need thereof a chimeric antibodycomprising a VH comprising an amino acid sequence of SEQ ID NO: 110 anda VL comprising an amino acid sequence of SEQ ID NO: 111 (e.g., antibodyxAb).

In another aspect, presented herein are methods for treating cancer in asubject, comprising administering to a subject in need thereof anantibody-drug conjugate described herein, or a pharmaceuticalcomposition comprising an antibody-drug conjugate described herein. Inanother aspect, presented herein are methods for treating cancer in asubject, comprising administering to a subject in need thereof abispecific antibody described herein, or a pharmaceutical compositioncomprising a bispecific antibody described herein. In another aspect,presented herein are methods for treating cancer in a subject,comprising administering to a subject in need thereof a polynucleotideor vector encoding an anti-MERTK antibody or antigen-binding fragmentthereof described herein, or a pharmaceutical composition comprising apolynucleotide(s) or vector(s) encoding an anti-MERTK antibody orantigen-binding fragment thereof described herein.

In a specific embodiment, presented herein are methods for treatingcancer in a subject, comprising administering to a subject in needthereof a pharmaceutical composition comprising an anti-MERTK antibodyor an antigen-binding fragment thereof described herein thatspecifically binds to MERTK (e.g., human MERTK), or an antibody-drugconjugate described herein.

In certain embodiments, the methods for treating cancer described hereininclude obtaining a tumor biopsy, tumor sample, or cancer cell samplefrom a subject and assessing the level of expression of MERTK (e.g.,human MERTK) using an assay described herein or known to one of skill inthe art. In some embodiments, the level of phosphorylation of MERTK(e.g., human MERTK) by cancer cells, a tumor sample or a tumor biopsyobtained from a subject is assessed using an assay described herein orknown to one of skill in the art. In some embodiments, the methods fortreating cancer described herein include obtaining a tumor biopsy, tumorsample, or cancer cell sample from a subject and assessing the level ofmutated MERTK (e.g., mutant human MERTK) using an assay described hereinor known in the art. Techniques known to one of skill in the art may beused to obtain a tumor biopsy or cancer cell sample. In someembodiments, a Western blot, an ELISA or flow cytometry is used toassess MERTK (e.g., human MERTK) expression levels. In certainembodiments, a Western blot or an ELISA is used to assess the level ofphosphorylation of MERTK (e.g., human MERTK). In some embodiments, ananti-MERTK antibody or antigen-binding fragment thereof describedherein, or an anti-MERTK known to one of skill in the art (e.g., ananti-MERTK antibody described in International Patent ApplicationPublication No. WO 2016106221) is used to measure MERTK (e.g., humanMERTK) levels. In some embodiments, a subject treated in accordance withthe methods described herein has cancer cells overexpressing MERTK,cancer cells expressing constitutively activated MERTK, or both.

In certain embodiments, the methods for treating cancer described hereininclude obtaining PMBCs from a subject, isolating macrophages andassessing the level of expression of MERTK (e.g., human MERTK) using anassay described herein or known to one of skill in the art. In someembodiments, PMBCs from a subject are obtained, macrophages areisolated, and the level of phosphorylation of expression of MERTK (e.g.,human MERTK), or level of mutated MERTK (e.g., mutated human MERTK), orboth by the macrophages is assessed using an assay described herein orknown to one of skill in the art. Techniques known to one of skill inthe art may be used to obtain PMBCs and isolate macrophages. In someembodiments, a Western blot, an ELISA or flow cytometry is used toassess MERTK (e.g., human MERTK) expression levels. In certainembodiments, a Western blot or an ELISA is used to assess the level ofphosphorylation of MERTK (e.g., human MERTK). In some embodiments, ananti-MERTK antibody or antigen-binding fragment thereof describedherein, or an anti-MERTK known to one of skill in the art (e.g., ananti-MERTK antibody described in International Patent ApplicationPublication No. WO 2016106221) is used to measure MERTK (e.g., humanMERTK) levels. In some embodiments, a subject treated in accordance withthe methods described herein has cancer cells overexpressing MERTK,cancer cells expressing constitutively activated MERTK, or both.

In certain embodiments, the level of expression of MERTK (e.g., humanMERTK) on cancer cells, macrophages, or both is assessed prior totreating a subject in accordance with the methods described herein. Incertain embodiments, the level of mutated MERTK (e.g. mutated humanMERTK) on cancer cells is assessed prior to treating a subject inaccordance with the methods described herein. In some embodiments, thelevel of phosphorylated MERTK (e.g., human MERTK) as well as the levelof mutated MERTK (e.g., mutated human MERTK) expressed by cancer cells,macrophages or both is assessed prior to treating a subject inaccordance with the methods described herein. In certain embodiments,the level of expression and phosphorylation of MERTK (e.g., human MERTK)on cancer cells, macrophages, or both is assessed prior to treating asubject in accordance with the methods described herein.

In certain embodiments, the level of expression of MERTK (e.g., humanMERTK) on cancer cells, macrophages, or both is assessed while treatinga subject in accordance with the methods described herein in order toassess the effectiveness of the treatment. In some embodiments, thelevel of phosphorylated MERTK (e.g., human MERTK) expressed by cancercells, macrophages or both is assessed while treating a subject inaccordance with the methods described herein in order to assess theeffectiveness of the treatment. In some embodiments, the level ofmutated MERTK (e.g., mutated human MERTK) is assessed while treating asubject in accordance with the methods described herein in order toassess the effectiveness of the treatment. In certain embodiments, thelevel of expression and phosphorylation of MERTK (e.g., human MERTK) aswell as the level of mutated MERTK (e.g., mutated human MERTK) on cancercells, macrophages, or both is assessed while treating a subject inaccordance with the methods described herein in order to assess theeffectiveness of the treatment.

In specific embodiments, the administration of an antibody orantigen-binding fragment, or the antibody-drug conjugate describedherein, or a pharmaceutical composition described herein to a subjectwith cancer achieves at least one, two, three, four or more of thefollowing effects: (i) the reduction or amelioration of the severity ofone or more symptoms of cancer; (ii) the reduction in the duration ofone or more symptoms associated with cancer; (iii) the prevention in therecurrence of a symptom associated with cancer; (iv) the reduction inhospitalization of a subject; (v) a reduction in hospitalization length;(vi) the increase in the survival of a subject; (vii) the enhancement orimprovement of the therapeutic effect of another therapy; (viii) theinhibition of the development or onset of one or more symptomsassociated with cancer; (ix) the reduction in the number of symptomsassociated with cancer; (x) improvement in quality of life as assessedby methods well known in the art; (x) inhibition of the recurrence of atumor; (xi) the regression of tumors and/or one or more symptomsassociated therewith; (xii) the inhibition of the progression of tumorsand/or one or more symptoms associated therewith; (xiii) a reduction inthe growth of a tumor; (xiv) a decrease in tumor size (e.g., volume ordiameter); (xv) a reduction in the formation of a newly formed tumor;(xvi) prevention, eradication, removal, or control of primary, regionaland/or metastatic tumors; (xvii) a decrease in the number or size ofmetastases; (xviii) a reduction in mortality; (xix) an increase inrelapse free survival; (xx) the size of the tumor is maintained and doesnot increase or increases by less than the increase of a tumor afteradministration of a standard therapy as measured by conventional methodsavailable to one of skill in the art, such as magnetic resonance imaging(MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray, and computedtomography (CT) scan, or a positron emission tomography (PET) scan;and/or (xxi) an increase in the length of remission in patients.

In some embodiments, a method of treating cancer as described hereinresults in one, two, three or more of the following effects: completeresponse, partial response, objective response, increase in overallsurvival, increase in disease free survival, increase in objectiveresponse rate, increase in time to progression, stable disease, increasein progression-free survival, increase in time-to-treatment failure, andimprovement or elimination of one or more symptoms of cancer. In aspecific embodiment, a method of treating cancer as described hereinresults in an increase in overall survival. In another specificembodiment, a method of treating cancer as described herein results inan increase in progression-free survival. In another specificembodiment, a method of treating cancer as described herein results inan increase in overall survival and an increase in progression-freesurvival.

In a specific embodiment, complete response has the meaning understoodby one of skill in the art. In a specific embodiment, complete responserefers to the disappearance of all signs of cancer in response totreatment. A complete response may not mean that the cancer is cured butthat patient is in remission. In a specific embodiment, colorectalcancer is in complete remission if clinically detectable disease is notdetected by known techniques such as radiographic studies, bone marrow,and biopsy or protein measurements.

In a specific embodiment, partial response has the meaning understood byone of skill in the art. For example, a partial response may refer to adecrease in the size of colorectal cancer in the human body in responseto the treatment. In a specific embodiment, a partial response refers toat least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decreasein all measurable tumor burden (e.g., the number of malignant cellspresent in the subject, or the measured bulk of tumor masses or thequantity of abnormal monoclonal protein) in the absence of new lesions.

In a specific embodiment, overall survival has the meaning understood byone of skill in the art. In a specific embodiment, overall survivalrefers to the length of time from either the date of the diagnosis orthe start of treatment for colorectal cancer, that the human subjectdiagnosed with colorectal cancer is still alive. Demonstration of astatistically significant improvement in overall survival can beconsidered to be clinically significant if the toxicity profile isacceptable, and has often supported new drug approval.

Several endpoints are typically based on tumor assessments. Theseendpoints include disease free survival (DFS), objective response rate(ORR), time to progression (TTP), progression-free survival (PFS), andtime-to-treatment failure (TTF). The collection and analysis of data onthese time-dependent endpoints are often based on indirect assessments,calculations, and estimates (e.g., tumor measurements).

In a specific embodiment, disease free survival (DFS) has the meaningunderstood by one of skill in the art. In a specific embodiment,disease-free survival may refer to the length of time after primarytreatment for colorectal cancer ends that the human subject surviveswithout any signs or symptoms of cancer. DFS can be an importantendpoint in situations where survival may be prolonged, making asurvival endpoint impractical. DFS can be a surrogate for clinicalbenefit or it can provide direct evidence of clinical benefit. Thisdetermination is typically based on the magnitude of the effect, itsrisk-benefit relationship, and the disease setting. The definition ofDFS can be complicated, particularly when deaths are noted without priortumor progression documentation. These events may be scored either asdisease recurrences or as censored events. Although all methods forstatistical analysis of deaths have some limitations, considering alldeaths (deaths from all causes) as recurrences can minimize bias. DFScan be overestimated using this definition, especially in patients whodie after a long period without observation. Bias can be introduced ifthe frequency of long-term follow-up visits is dissimilar between thestudy arms or if dropouts are not random because of toxicity.

In a specific embodiment, objective response rate has the meaningunderstood by one of skill in the art. In one embodiment, an objectiveresponse rate is defined as the proportion of patients with tumor sizereduction of a predefined amount and for a minimum time period. Responseduration maybe measured from the time of initial response untildocumented tumor progression. Generally, the FDA has defined ORR as thesum of partial responses plus complete responses. When defined in thismanner, ORR is a direct measure of drug antitumor activity, which can beevaluated in a single-arm study. If available, standardized criteriashould be used to ascertain response. A variety of response criteriahave been considered appropriate (e.g., RECIST1.1 criteria) (See e.g.,Eisenhower et al., 2009, European J. of Cancer, 45: 228-247)). Thesignificance of ORR is assessed by its magnitude and duration, and thepercentage of complete responses (no detectable evidence of tumor).

In a specific embodiment, time to progression (TTP) has the meaningunderstood by one of skill in the art. In a specific embodiment, time toprogression refers to the length of time from the date of diagnosis orstart of treatment for colorectal cancer until the cancer gets worse orspreads to other parts of the human body.

In a specific embodiment, TTP is the time from randomization untilobjective tumor progression; TTP does not include deaths.

In a specific embodiment, progression free survival (PFS) has themeaning understood by one of skill in the art. In a specific embodiment,PFS refers to the length of time during and after treatment ofcolorectal cancer that the human patient lives with the cancer but itdoes not get worse. In a specific embodiment, PFS is defined as the timefrom randomization until objective tumor progression or death. PFS mayinclude deaths and thus can be a better correlate to overall survival.

In a specific embodiment, time-to-treatment failure (TTF) has themeaning understood by one of skill in the art. In a specific embodiment,TTF is composite endpoint measuring time from randomization todiscontinuation of treatment for any reason, including diseaseprogression, treatment toxicity, and death.

In a specific embodiment, stable disease refers to colorectal cancerthat is neither decreasing or increasing in extent or severity.

In a specific embodiment, the RECIST 1.1 criteria is used to measure howwell a human subject responds to the treatment methods described herein.

In some embodiments, the cancer treated in accordance with the methodsdescribed herein is a cancer of the head and neck, lung, breast, bone,ovary, stomach, pancreas, larynx, esophagus, testes, liver, gastric,parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,kidney, bladder, prostate or thyroid. In some embodiments, the cancer isa sarcoma, squamous cell carcinoma, melanoma, glioma, glioblastoma,neuroblastoma, non-small cell lung carcinoma (NSCLC), head and neckcancer, colorectal cancer, or Kaposi's sarcomas. In some embodiments,the cancer is a leukemia or lymphoma. In some embodiments, the cancer ismultiple myeloma. In some embodiments, the cancer treated in accordancewith the methods is metastatic.

In a specific embodiment, the cancer treated in accordance with themethods described herein is breast cancer. In a particular embodiment,the cancer treated in accordance with the methods described herein istriple-negative breast cancer. Triple-negative breast cancer refers toany tumor or cell derived from or growing in breast tissue that does notexpress the genes Her2 (also known as Neu), Estrogen Receptor (alsoknown as ER) or Progesterone Receptor (also known as PR).

In another specific embodiment, the cancer treated in accordance withthe methods described herein is acute myelogenous leukemia. In yetanother specific embodiment, the cancer treated in accordance with themethods described herein is acute lymphocytic leukemia.

In certain embodiments, the cancerous cells of the cancer treated inaccordance with the methods described herein overexpress MERTK. In aspecific embodiment, MERTK is constitutively active in the cancerouscells of the cancer treated in accordance with the methods describedherein.

In certain embodiments, the cancer treated in accordance with themethods described herein is associated with constitutively active MERTK.In another specific embodiment, the cancer treated in accordance withthe methods described herein is associated with overexpression of MERTK.In another specific embodiment, the cancer treated in accordance withthe methods described herein is associated with overexpression of MERTKwhich is constitutively active.

As used herein, the terms “subject” and “patient” are usedinterchangeably. In some embodiments, the subject is a mammal such as aprimate (e.g., monkey or human), most preferably a human.

In a specific embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof that specifically binds to MERTK (e.g., human MERTK),(e.g., z10, z11, z13 or xAb antibodies), or an antibody-drug conjugatedescribed herein are administered to a subject. In certain embodiments,two or more different antibodies or antigen-binding fragments thereofthat specifically binds MERTK (e.g., human MERTK), described herein areadministered to a subject. In certain embodiments, an anti-MERTKantibody or antigen-binding fragments thereof described herein thatspecifically binds MERTK (e.g., human MERTK), described herein areadministered to a subject in combination with one or more othertherapies (See Section 5.4.2, InJfra.).

In certain embodiments, two or more different antibody-drug conjugatesdescribed herein are administered to a subject. In some embodiments, anantibody-drug conjugate described herein is administered to a subject incombination with one or more other therapies. In some embodiments, ananti-MERTK antibody (e.g., a humanized antibody) or an antigen-bindingfragment thereof, or an antibody-drug conjugate described herein isadministered to a subject in combination with one or more othertherapies (See Section 5.4.2, Infra.). In specific embodiments, the oneor more other therapies are anti-cancer therapies.

5.6.1.2 Routes of Administration and Dosage

An anti-MERTK antibody or antigen-binding fragment thereof, a chimericanti-MERTK antibody or antigen-binding fragment thereof, or anantibody-drug conjugate described herein, or a pharmaceuticalcomposition described herein may be delivered to a subject by a varietyof routes. These include, but are not limited to, parenteral,intranasal, intratracheal, oral, intradermal, topical, intramuscular,intraperitoneal, transdermal, intravenous, intratumoral, conjunctival,intratumoral and subcutaneous routes. Pulmonary administration can alsobe employed, e.g., by use of an inhaler or nebulizer, and formulationwith an aerosolizing agent for use as a spray. In one embodiment, ananti-MERTK antibody or antigen-binding fragment thereof or anantibody-drug conjugate described herein, or a pharmaceuticalcomposition described herein is administered parenterally to a subject.In a specific embodiment, said parenteral administration is intravenous,intramuscular, or subcutaneous.

The amount of an anti-MERTK antibody or antigen-binding fragmentthereof, or an antibody-drug conjugate, or pharmaceutical compositionwhich will be effective in the treatment and/or prevention of acondition will depend on the nature of the disease, and can bedetermined by standard clinical techniques.

The precise dose of an anti-MERTK antibody or antigen-binding fragmentthereof, or the antibody-drug conjugate to be employed in apharmaceutical composition will also depend on the route ofadministration, and the type of cancer, and should be decided accordingto the judgment of the practitioner and each subject's circumstances.For example, effective doses may also vary depending upon means ofadministration, target site, physiological state of the patient(including age, body weight and health), whether the patient is human oranimal, other medications administered, or whether treatment isprophylactic or therapeutic. Treatment dosages are optimally titrated tooptimize safety and efficacy.

In certain embodiments, an in vitro assay is employed to help identifyoptimal dosage ranges. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

For an antibody (or an antigen-binding fragment thereof) or an antibodydrug conjugates, the dosage ranges from about 0.0001 to 100 mg/kg, andmore usually 0.01 to 15 mg/kg, of the patient body weight. For example,dosages can be 1 mg/kg body weight, 10 mg/kg body weight, or within therange of 1-10 mg/kg or in other words, 70 mg or 700 mg or within therange of 70-700 mg, respectively, for a 70 kg patient. In someembodiments, the dosage administered to the patient is about 1 mg/kg toabout 20 mg/kg of the patient's body weight.

An exemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months for a period of oneyear or over several years, or over several year-intervals. In somemethods, two or more antibodies or antigen-binding fragments thereofwith different binding specificities are administered simultaneously toa subject. An anti-MERTK antibody or antigen-binding fragment thereof,or an antibody-drug conjugate is usually administered on multipleoccasions. Intervals between single dosages can be weekly, monthly,every 3 months, every 6 months or yearly.

5. 6. 1. 3 Combination Therapies

In a specific aspect, the methods for treating cancer described hereinfurther comprise administering one or more other therapies, such asanti-cancer therapies (e.g., surgery, radiation, chemotherapy, or anadditional therapeutic agent). In a specific embodiment, the methodsprovided herein for treating cancer in a subject, comprisingadministering to a subject in need thereof a pharmaceutical compositioncomprising an anti-MERTK antibody or an antigen-binding fragmentthereof, or an antibody-drug conjugate described herein, furthercomprise administering to the subject one or more additional therapeuticagents. In a specific embodiment, the additional therapeutic agent isfor treating the cancer. In a specific embodiment, the additionaltherapeutic agent is for treating any side effects of treatment with ananti-MERTK antibody or antigen binding fragment thereof, or anantibody-drug conjugate described herein.

In specific embodiments, the additional agent is an agent used to treatbreast cancer, an agent used to treat melanoma, an immunotherapy, or anangiogenesis inhibitor.

In a specific embodiment, the additional therapeutic agent is an agentused to treat breast cancer that is selected from the group consistingof Tamoxifen, Raloxifene, Paclitaxel (TAXOL®), Cyclophosphamide,Docetaxel, Vinblastine, Fluorouracil, Everolimus, Trastuzumab,Trastuzumab-Emtansine, Pertuzumab, and Lapatinib Ditosylate.

In a specific embodiment, the additional therapeutic agent is an agentused to treat melanoma that is selected from the group consisting of aBRAF inhibitor, a MEK inhibitor, and Dacarbazine.

In a specific embodiment, the additional therapeutic agent is an agentthat blocks immune checkpoint signaling. In specific embodiments, theadditional therapeutic agent is an anti-CTLA-4 blocking antibody, ananti-PD-1 blocking antibody, or an anti-PD-L1 blocking antibody.

In a specific embodiment, the additional therapeutic agent is anangiogenesis inhibitor that is selected from the group consisting of aVEGF inhibitor, a VEGFR2 inhibitor, Sunitinib, and Sorafenib.

In other embodiments, the additional therapeutic agent is an agentlisted in Table 32.

TABLE 32 Additional Therapeutic Agents for Use in Combination Therapywith MERTK Antibodies or Antigen-Binding Fragments Thereof Alkylatingagents Busulfan Chlorambucil dacarbazine procarbazine ifosfamidealtretamine hexamethylmelamine estramustine phosphate thiotepamechlorethamine dacarbazine streptozocin lomustine temozolomidecyclophosphamide Semustine Platinum agents spiroplatin lobaplatin(Aeterna) tetraplatin satraplatin (Johnson Matthey) ormaplatin BBR-3464(Hoffmann-La Roche) iproplatin SM-11355 (Sumitomo) ZD-0473 (AnorMED)AP-5280 (Access) oxaliplatin cisplatin carboplatin Antimetabolitesazacytidine trimetrexate Floxuridine deoxycoformycin2-chlorodeoxyadenosine pentostatin 6-mercaptopurine hydroxyurea6-thioguanine decitabine (SuperGen) cytarabine clofarabine (Bioenvision)2-fluorodeoxy cytidine irofulven (MGI Pharma) methotrexate DMDC(Hoffmann-La Roche) tomudex ethynylcytidine (Taiho) fludarabinegemcitabine raltitrexed capecitabine Topoisomerase amsacrine exatecanmesylate (Daiichi) inhibitors epirubicin quinamed (ChemGenex) etoposidegimatecan (Sigma-Tau) teniposide or mitoxantrone diflomotecan(Beaufour-Ipsen) 7-ethyl-10-hydroxy- TAS-103 (Taiho) camptothecindexrazoxanet (TopoTarget) elsamitrucin (Spectrum) pixantrone(Novuspharma) J-107088 (Merck & Co) rebeccamycin analogue BNP-1350(BioNumerik) (Exelixis) BBR-3576 (Novuspharma) CKD-602 (Chong Kun Dang)rubitecan (SuperGen) KW-2170 (Kyowa Hakko) irinotecan (CPT-11)hydroxycamptothecin (SN-38) topotecan Antitumor valrubicin azonafideantibiotics therarubicin anthrapyrazole idarubicin oxantrazolerubidazone losoxantrone plicamycin MEN-10755 (Menarini) porfiromycinGPX-100 (Gem Pharmaceuticals) mitoxantrone (novantrone) Epirubicinamonafide mitoxantrone doxorubicin Antimitotic colchicine E7010 (Abbott)agents vinblastine PG-TXL (Cell Therapeutics) vindesine IDN 5109 (Bayer)dolastatin 10 (NCI) A 105972 (Abbott) rhizoxin (Fujisawa) A 204197(Abbott) mivobulin (Warner-Lambert) LU 223651 (BASF) cemadotin (BASF) D24851 (ASTAMedica) RPR 109881A (Aventis) ER-86526 (Eisai) TXD 258(Aventis) combretastatin A4 (BMS) epothilone B (Novartis)isohomohalichondrin-B (PharmaMar) T 900607 (Tularik) ZD 6126(AstraZeneca) T 138067 (Tularik) AZ10992 (Asahi) cryptophycin 52 (EliLilly) IDN-5109 (Indena) vinflunine (Fabre) AVLB (Prescient NeuroPharma)auristatin PE (Teikoku azaepothilone B (BMS) Hormone) BMS 247550 (BMS)BNP-7787 (BioNumerik) BMS 184476 (BMS) CA-4 prodrug (OXiGENE) BMS 188797(BMS) dolastatin-10 (NIH) taxoprexin (Protarga) CA-4 (OXiGENE) SB 408075docetaxel (GlaxoSmithKline) Vinorelbine vincristine Trichostatin Apaclitaxel Aromatase aminoglutethimide YM-511 (Yamanouchi) inhibitorsatamestane (BioMedicines) formestane letrozole exemestane anastrazoleThymidylate pemetrexed (Eli Lilly) nolatrexed (Eximias) synthaseinhibitors ZD-9331 (BTG) CoFactor ™ (BioKeys) DNA antagoniststrabectedin (PharmaMar) edotreotide (Novartis) glufosfamide (Baxtermafosfamide (Baxter International) International) albumin + 32P (Isotopeapaziquone (Spectrum Solutions) Pharmaceuticals) thymectacin(NewBiotics) O6 benzyl guanine (Paligent) Farnesyltransferase arglabin(NuOncology Labs) tipifarnib (Johnson & Johnson) inhibitors lonafarnib(Schering-Plough) perillyl alcohol (DOR BioPharma) BAY-43-9006 (Bayer)Pump inhibitors CBT-1 (CBA Pharma) zosuquidar trihydrochloride (EliLilly) tariquidar (Xenova) biricodar dicitrate (Vertex) MS-209 (ScheringAG) Histone tacedinaline (Pfizer) pivaloyloxymethyl butyrate (Titan)acetyltransferase SAHA (Aton Pharma) depsipeptide (Fujisawa) inhibitorsMS-275 (Schering AG) Metalloproteinase Neovastat (Aeterna CMT-3(CollaGenex) inhibitors Laboratories) marimastat (British Biotech)BMS-275291 (Celltech) Ribonucleoside gallium maltolate (Titan)tezacitabine (Aventis) reductase inhibitors triapine (Vion) didox(Molecules for Health) TNF alpha virulizin (Lorus Therapeutics) revimid(Celgene) agonists/antagonists CDC-394 (Celgene) Endothelin A atrasentan(Abbott) YM-598 (Yamanouchi) receptor antagonist ZD-4054 (AstraZeneca)Retinoic acid fenretinide (Johnson & alitretinoin (Ligand) Johnson)receptor agonists LGD-1550 (Ligand) Immunomodulators interferon dexosometherapy (Anosys) oncophage (Antigenics) pentrix (Australian CancerTechnology) GMK (Progenics) ISF-154 (Tragen) adenocarcinoma vaccinecancer vaccine (Intercell) (Biomira) CTP-37 (AVI BioPharma) norelin(Biostar) IRX-2 (Immuno-Rx) BLP-25 (Biomira) PEP-005 (Peplin Biotech)MGV (Progenics) synchrovax vaccines (CTL β-alethine (Dovetail) Immuno)melanoma vaccine (CTL CLL therapy (Vasogen) Immuno) p21 RAS vaccine(GemVax) Ipilimumab (BMS), MAGE-A3 (GSK) CM-10 (cCam Biotherapeutics)nivolumab (BMS) MPDL3280A (Genentech) abatacept (BMS) pidilizumab(CureTech) lambrolizumab (Merck) AMP-224 (GSK) MEDI-4736 (AstraZeneca)Pembrolizumab Avelumab Anti-Lag-3 therapies Anti-GITR therapiesAnti-TFG-beta therapies OX40 agonists CAR-T Cell Therapies KymriahJCar017 Yescarta RGX-014 RGX-202 M7824 (bintrafusp alfa) Hormonal andestrogens dexamethasone anti hormonal conjugated estrogens Prednisoneagents ethinyl estradiol Methylprednisolone chlortrianisen Prednisoloneidenestrol Aminoglutethimide hydroxyprogesterone caproate Leuprolidemedroxyprogesterone Octreotide testosterone Mitotane testosteronepropionate; P-04 (Novogen) fluoxymesterone methyltestosterone2-methoxyestradiol (EntreMed) diethylstilbestrol arzoxifene (Eli Lilly)megestrol Tamoxifen bicalutamide Toremofine flutamide Goserelinnilutamide Leuporelin Bicalutamide Photodynamic talaporfin (LightSciences) Pd-bacteriopheophorbide (Yeda) agents Theralux(Theratechnologies) lutetium texaphyrin (Pharmacyclics) motexafingadolinium hypericin (Pharmacyclics) Kinase Inhibitors imatinib(Novartis) EKB-569 (Wyeth) leflunomide kahalide F (PharmaMar)(Sugen/Pharmacia) ZD1839 (AstraZeneca) CEP-701 (Cephalon) erlotinib(Oncogene Science) CEP-751 (Cephalon) canertinib (Pfizer) MLN518(Millenium) squalamine (Genaera) PKC412 (Novartis) SU5416 (Pharmacia)Phenoxodiol (Novogen) SU6668 (Pharmacia) C225 (ImClone) ZD4190(AstraZeneca) rhu-Mab (Genentech) ZD6474 (AstraZeneca) MDX-H210(Medarex) vatalanib (Novartis) 2C4 (Genentech) PKI166 (Novartis) MDX-447(Medarex) GW2016 (GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth)IMC-1C11 (ImClone) trastuzumab (Genentech) Tyrphostins OSI-774(Tarceva ™) Gefitinib (Iressa) CI-1033 (Pfizer) PTK787 (Novartis)SU11248 (Pharmacia) EMD 72000 (Merck) RH3 (York Medical) EmodinGenistein Radicinol Radicinol Vemurafenib (B-Raf enzyme inhibitor,Daiichi Sankyo) Met-MAb (Roche) trametinib (GSK) Additional AgentsSR-27897 (CCK A inhibitor, ceflatonin (apoptosis promotor,Sanofi-Synthelabo) ChemGenex) tocladesine (cyclic AMP BCX-1777 (PNPinhibitor, BioCryst) agonist, Ribapharm) alvocidib (CDK inhibitor,ranpirnase (ribonuclease stimulant, Aventis) Alfacell) CV-247 (COX-2inhibitor, galarubicin (RNA synthesis inhibitor, Ivy Medical) Dong-A)P54 (COX-2 inhibitor, tirapazamine (reducing agent, SRI Phytopharm)International) CapCell ™ (CYP450 N-acetylcysteine (reducing agent,stimulant, Bavarian Nordic) Zambon) GCS-100 (gal3 antagonist,R-flurbiprofen (NF-kappaB inhibitor, GlycoGenesys) Encore) G17DTimmunogen (gastrin 3CPA (NF-kappaB inhibitor, Active inhibitor, Aphton)Biotech) efaproxiral (oxygenator, Allos seocalcitol (vitamin D receptorTherapeutics) agonist, Leo) PI-88 (heparanase inhibitor, 131-I-TM-601(DNA antagonist, Progen) TransMolecular) tesmilifene (histamineeflornithine (ODC inhibitor, ILEX antagonist, YM BioSciences) Oncology)histamine (histamine H2 minodronic acid (osteoclast inhibitor, receptoragonist, Maxim) Yamanouchi) tiazofurin (IMPDH inhibitor, indisulam (p53stimulant, Eisai) Ribapharm) cilengitide (integrin aplidine (PPTinhibitor, PharmaMar) antagonist, Merck KGaA) SR-31747 (IL-1 antagonist,gemtuzumab (CD33 antibody, Wyeth Sanofi-Synthelabo) Ayerst) CCI-779(mTOR kinase PG2 (hematopoiesis enhancer, inhibitor, Wyeth)Pharmagenesis) exisulind (PDE V inhibitor, Immunol ™ (triclosan oralrinse, Cell Pathways) Endo) CP-461 (PDE V inhibitor, triacetyluridine(uridine prodrug, Cell Pathways) Wellstat) AG-2037 (GART inhibitor,SN-4071 (sarcoma agent, Signature Pfizer) BioScience) WX-UK1(plasminogen TransMID-107 ™ (immunotoxin, KS activator inhibitor, Wilex)Biomedix) PBI-1402 (PMN stimulant, PCK-3145 (apoptosis promotor,ProMetic LifeSciences) Procyon) bortezomib (proteasome doranidazole(apoptosis promotor, inhibitor, Millennium) Pola) SRL-172 (T cellstimulant, CHS-828 (cytotoxic agent, Leo) SR Pharma) TLK-286(glutathione S trans-retinoic acid (differentiator, transferaseinhibitor, Telik) NIH) PT-100 (growth factor MX6 (apoptosis promotor,MAXIA) agonist, Point Therapeutics) midostaurin (PKC inhibitor, apomine(apoptosis promotor, ILEX Novartis) Oncology) bryostatin-1 (PKCstimulant, urocidin (apoptosis promotor, GPC Biotech) Bioniche) CDA-II(apoptosis promotor, Ro-31-7453 (apoptosis promotor, La Everlife) Roche)SDX-101 (apoptosis brostallicin (apoptosis promotor, promotor, Salmedix)Pharmacia) rituximab (CD20 antibody, β-lapachone Genentech carmustinegelonin Mitoxantrone cafestol Bleomycin kahweol Absinthin caffeic acidChrysophanic acid Tyrphostin AG Cesium oxides PD-1 inhibitors BRAFinhibitors, CTLA-4 inhibitors PDL1 inhibitors sorafenib MEK inhibitorsBRAF inhibitors bevacizumab angiogenesis inhibitors dabrafenib

In a specific embodiment, a method for treating cancer described hereincomprising administering an anti-MERTK antibody or antigen-bindingfragment thereof to a subject, may further comprise administering ananti-PD1 antibody (e.g., pembrolizumab, cemiplimab, pidilizumab, ornivolumab), an anti-PD-L1 antibody (e.g., atezolizumab avelumab,durvaniab, BMS-936552, or CK-301), an anti-LAG3 antibody, agonisticanti-GITR antibody, an anti-TGF-beta antibody, an agonistic anti-OX 40antibody, a CAR-T therapy (e.g., Kymriah, JCar017, or Yescarta),RGX-104, or RGX-202.

An anti-MERTK antibody or antigen-binding fragment thereof or anantibody-drug conjugate described herein can be administered with anadditional therapeutic agent concurrently or sequentially (before and/orafter). The antibody or antigen binding fragment thereof and theadditional therapeutic agent, or the antibody-drug conjugate and theadditional therapeutic agent can be administered in the same ordifferent compositions, and by the same or different routes ofadministration. A first therapy (which is an anti-MERTK antibody orantigen-binding fragment thereof or an antibody-drug conjugate describedherein, or the additional therapeutic agent) can be administered priorto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of the second therapy (the anti-MERTK antibody orantigen-binding fragment thereof or the antibody-drug conjugatedescribed herein, or the additional therapeutic agent) to a subject withcancer. In certain embodiments, an additional therapeutic agentadministered to a subject in combination with an anti-MERTK antibody orantigen-binding fragment thereof or an antibody-drug conjugate isadministered in the same composition (pharmaceutical composition). Inother embodiments, an additional therapeutic agent administered incombination with an anti-MERTK antibody or antigen-binding fragmentthereof or an antibody-drug conjugate is administered to a subject in adifferent composition than the anti-MERTK antibody or antigen-bindingfragment thereof or the antibody-drug conjugate (e.g., two or morepharmaceutical compositions are used).

5.7 Kits

Also provided herein are kits comprising one or more antibodies orantibody-drug conjugates described herein, or antigen-binding fragmentsthereof. In a specific embodiment, provided herein is a pharmaceuticalpack or kit comprising one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions described herein,such as one or more antibodies or an antigen-binding fragment thereof orone or more antibody-drug conjugates described herein. In someembodiments, the kits contain a pharmaceutical composition describedherein and a prophylactic or therapeutic agent.

Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, a dosage form, and/orinstructions for use thereof. In certain embodiments, the instructionsincluded with the kit provide guidance with respect to the dosageamounts and/or dosing regimens for administration of the pharmaceuticalcomposition(s).

Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, packets, sachets, tubes, inhalers,pumps, bags, vials, containers, syringes and any packaging materialsuitable for a selected pharmaceutical composition and intended mode ofadministration and treatment.

Kits provided herein can further include devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, needle-less injectors, drip bags, patchesand inhalers.

Kits provided herein can further include pharmaceutically acceptablevehicles that can be used to administer the ingredients. For example, ifan ingredient is provided in a solid form that must be reconstituted forparenteral administration, the kit can comprise a sealed container of asuitable vehicle in which the ingredient can be dissolved to form aparticulate-free sterile solution that is suitable for parenteraladministration or can be reconstituted as a suspension for oraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: aqueous vehicles including, but notlimited to, Water for Injection USP, Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,and Lactated Ringer's Injection; water-miscible vehicles including, butnot limited to, ethyl alcohol, polyethylene glycol, and polypropyleneglycol; and non-aqueous vehicles including, but not limited to, cornoil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropylmyristate, and benzyl benzoate.

In some embodiments, a kit described herein includes a reagent (e.g., ananti-MERTK antibody or antigen-binding fragment thereof describedherein, or an anti-MERTK antibody known to one of skill in the art(e.g., an anti-MERTK antibody described in International PatentApplication Publication No. WO 2016106221)) to measure MERTK (e.g.,human MERTK) levels on cells (e.g., cancer cells, macrophages or both)from a subject prior to and/or while treating the subject in accordancewith the methods described herein. In certain embodiments, a kitdescribed herein includes a reagent(s) to measure the level ofphosphorylated MERTK (e.g., human MERTK) by cells (e.g., cancer cells,macrophages or both) from a subject to be treated in accordance with themethods described herein. In certain embodiments, a kit described hereinincludes a reagent(s) to measure the level of mutated MERTK (e.g., humanMERTK) by cells (e.g., cancer cells, macrophages or both) from a subjectto be treated in accordance with the methods described herein. In someembodiments, a kit includes information that guides aclinician/physician regarding the readout of one, two, or all of thefollowing: MERTK (e.g., human MERTK) expression level, level ofphosphorylated MERTK (e.g., human MERTK), levels of mutated MERTK (e.g.,human MERTK).

The following examples are offered by way of illustration and not by wayof limitation.

6. EXAMPLES 6.1. Example 1: Generation of a Humanized, High-Affinity,Specific Antibody Specific to Human MERTK

This example describes a monoclonal antibody that leads to degradationof MERTK from the cell surface by internalization.

6.1.1. Humanization, Expression and Purification of Candidate Antibodies

To generate humanized anti-MERTK, the heavy chain and light chainframework regions of a subclone of the M6 antibody, a murine anti-MERTKantibody disclosed in U.S. Pat. No. 10,221,248 were replaced by humanheavy chain and light chain framework regions, respectively, optionallywith one or more back mutations. Combinations of heavy chains (S24, S25,S26, S27, S28, S29, S30, S31) and light chains (S32, S33) produced 16humanized antibodies in total. The humanized antibody candidates wereexpressed in HEK-293 cells or 293 F cells and expression was confirmedby gel electrophoresis (data not shown). A band was present at theexpected size of approximately 150 kD for humanized antibodies z1 thoughz16, as well as xAb (a chimeric antibody, Lane 17) and two hybridomalines (HyAb1 and HyAb2, Lanes 18 and 19). Binding affinity of thecandidate antibodies to human MERTK was measured by SPR. SPR results aresummarized in FIG. 1. Off-rate (K_(d)) values of less than 1×10⁻⁶ inFIG. 1 indicate that the Kd is below the detection limit of theinstrument. With respect to the variable light chain region, the backmutation of the fourth residue (serine, S) in framework region 3 (underthe Exemplary definition) of the variable light chain region to asparticacid (D) was found to be associated with an increase in binding affinityof the humanized antibodies to human MERTK. With respect to the variableheavy chain region, the back mutation of the₂₇th residue (Tyrosine, T,second to last residue) was found to be associated with a lower bindingaffinity of the humanized antibodies to human MERTK. As shown in FIG. 2,the combination of the variable region of the heavy chain with theframework regions for z10, z11 and z13 and the variable region of thelight chain with the framework region for z10, z11 and z13, which areidentical, resulted in humanized antibodies with a higher affinity thanwhen the variable region heavy chain with the framework region for z10,z11 and z13 were combined with a variable region light chain with theframework regions in Table 33 infra.

Table 34, infra, provides the avidity of the z10, z11 and z13 antibodiesusing SPR. Table 34, infra, provides the affinity of the z10 antibodyusing SPR and the ELISA cell-based assays. With respect to avidity andaffinity measurements using SPR, the protocols below were used. The z10antibody used in this study comprises the heavy and light chains setforth in Tables 16. The z11 antibody used in this study comprises theheavy and light chains set forth in Tables 17. The z13 antibody used inthis study comprises the heavy and light chains set forth in Tables 18.

Affinity Measurement of z10 to Human MERTK Using SPR

A CM5 sensor chip was activated for 420 s with a mixture of 400 mM EDCand 100 mM NHS. 30 μg/mL of Anti-human Fc IgG antibody (Jackson,109-005-098) in 10 mM NaAc (pH 4.5) was then injected for 420 s at aflow rate of 10 μL/min. The chip was deactivated by 1 Methanolamine-HCl. 5 μg/mL z10 antibody in running buffer (1 xHBS-EP+)was injected at a flow rate of 10 μL/min for 15 s. 6 concentrations(2.78, 5.56, 11.11, 22.22, 44.44 and 88.88 nM) of the extracellularMERTK domain (W3725-hPro1, Sino Biological, 10298-HCCH) and runningbuffer were injected at a flow rate of 30 μL/min for an associationphase of 180 s, followed by 600 s dissociation. 10 mM glycine pH 1.5 asregeneration buffer was injected following every dissociation phase. Thechip was regenerated with 10 mM glycine pH 1.5. One surface channel(without captured ligand) was used as control surface for referencesubtraction. Final data of each interaction was deducted from referencechannel and buffer channel data. The experimental data of z10 antibodybinding to W3725-hPro1 was fitted by 1:1 binding mode. A molecularweight of 54 kDa was used to calculate the molar concentration of theanalyte.

Avidity Measurement of z10, z11 and z13 to Human MERTK Using SPR

The activator was prepared with a mixture of 400 mM EDC and 100 mM NHSimmediately prior to injection. The CM5 sensor chip was activated for420 s with the mixture. 5 g/mL rhMer/Fc Chimera (R&D Systems, 8910MR) in10 mM NaAc (pH 4.5) was then injected for 30 s at a flow rate of 10μL/min. The chip was deactivated by 1 M ethanolamine-HCl. Z10, z11 andz13 antibodies were diluted with running buffer (1 xHBS-EP+). 8concentrations (0.3125, 0.625, 1.25, 2.5, 5, 10, 20 and 40 nM) of theantibodies and running buffer were injected at a flow rate of 30 μL/minfor an association phase of 240 s, followed by 4800 s dissociation. 10mM glycine pH 1.5 as regeneration buffer was injected following everydissociation phase. The chip was regenerated with 10 mM glycine pH 1.5.One surface channel without capturing ligand was used as control surfacefor reference subtraction. Final data of each interaction was deductedfrom reference channel and buffer channel data. The experimental data ofthe antibodies binding to rhMer/Fc Chimera was fitted by 1:1 bindingmode. The curves of 40 nM and 0.3125 nM antibodies were removed to allowa better fit. A molecular weight of 150 kDa was used to calculate themolar concentration of the antibodies.

TABLE 33 S32 heavy chain framework regions FR1 FR2 FR3 FR4 z32DIQMTQTPSFLSA WYQQKPGK GVPSRFSGSGSGTDFTL FGQGTK SVGDRVTITC APKLLIYTISSLQPEDFATYYC LEIK

TABLE 34 Avidity and affinity of the z10, z11 and z13 antibodies usingSPR Antibody Binding type Method Value Comment Z10 Avidity SPR KD = 5.7410⁻¹² M KD ELISA 1.46 nM KD Affinity SPR   3 nM KD Z11 Avidity SPR KD =2.99 10⁻¹² M KD Z13 SPR KD = 3.68 10⁻¹² M KD

Based on their high affinity for MERTK, z10, z11 and z13 were selectedfor further studies. Antibodies were purified using protein A andanalyzed by size-exclusion high performance liquid chromatography (SECHPLC). FIG. 2 shows a summary of the purity analysis for the fourantibodies. SDS-PAGE was used to characterize the four antibodies. Asexpected, 150 kDa, 50 kDa and 25 kDa bands were visible on the gel,suggesting the antibodies are normal.

6.2. Example 2: Activity of a Humanized Anti-MERTK Antibody

This example demonstrates that a particular humanized antibodyspecifically binds to human MERTK and does not bind to the extracellulardomain of human Axl, human Tyro3, or murine MERTK. This example alsodemonstrates that a humanized antibody that specifically binds to humanMERTK blocks Gas6-induced activation and inhibits colony formation ofcancer cells in vitro. Further, the example demonstrates that ahumanized antibody that specifically binds to human MERTK, reduces theexpression of human MERTK on the surface of cancer cells and macrophagesby inducing internalization and degradation of human MERTK.

6.2.1. Materials and Methods

Cell Lines, Antibodies and Reagents

SKMEL5 cells were obtained from ATCC (atcc, HTB-70) and cultured inEagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS,penicillin and streptomycin at 37° C. with 5% CO2. Antibodies for flowcytometry were obtained from the following sources: anti-MERTK(Biolegend, 367603), anti-CD86 (ThermoFisher, 12-0869-41), andanti-CD163 (ThermoFisher, 17-1639-41). Fixable Viability Dye (eFluor780) was obtained from Thermofisher. Antibodies for Western blots wereobtained from following sources: anti-MERTK (abcam, ab52968), anti-AKT(abcam, ab32505), anti-AKT1 phospho S473 (abcam, ab81283), anti-β-Actin(Sigma, A5316), and anti-α-Tubulin (Sigma, T5168).

Cell Surface Binding Assay

Z10 and human IgG control (R&D Systems, 1-001-A) were labeled with ZenonAllophycocyanin (APC) Human IgG Labeling Kit (ThermoFisher, Z25451)according to the manufacturer's instructions. SKMEL5 cells wereharvested with trypsin, washed twice with PBS and strained through a 70m filter. 50,000 cells were resuspended in 60 μL FACS buffer (2% BSA, 10mM EDTA, 25 mM HEPES, 0.1% sodium azide in Ca/Mg-free PBS) containing10% human AB serum (Sigma, H4522). The cells were stained with DAPI (500ng/mL) and APC-labeled Z10 or IgG control at concentrations ranging from0.002 to 13.5 μg/mL (0.13 to 90 nM) for 20 min on ice, then washed twicewith FACS buffer, and subjected to flow cytometer analysis (BD LSRFortessa). The APC signals from live single cells were analyzed inFlowJo and the MFIs were plotted with GraphPad Prism. Single colorcontrol samples were used to create a compensation matrix.

Internalization Assay

Z10 and human IgG control (R&D Systems) were labeled with pHrodo Red, apH-sensitive dye, using pHrodo Red Microscale Labeling Kit(ThermoFisher, Cat #) according to the manufacturer's instructions.Internalization of antibody was determined by flow cytometry detectingpHrodo fluorescence, which is minimal at neutral pH and maximal inacidic environments, such as the lysosomes. One million SKMEL5 wereplated on 6-well plates and incubated with 1 μg/mL pHrodo-labeled Z10 orIgG control antibody for 1, 3, 6 or 24 hrs. The cells were harvestedwith Cell Dissociation Buffer (ThermoFisher), washed twice, strainedthrough a 70 m filter, and resuspended in 200 μL FACS buffer (2% BSA, 10mM EDTA, 25 mM HEPES, 0.1% sodium azide in Ca/Mg-free PBS) containing10% human AB serum. Cells were sequentially stained withBV421-conjugated antibodies against MERTK for 20 min on ice, and thenstained with Fixable Viability Dye (eFluor 780) for 20 min on ice. 5,000event multispectral images were acquired for each sample by ImageStreamImaging Flow Cytometer (Aminis). Single color control samples were usedto create a compensation matrix, and resulting compensated data wereanalyzed by IDEA software (Amnis) and GraphPad Prism.

PBMC-Derived Cell Culture and Macrophage Differentiation

Peripheral blood mononuclear cells (PBMCs) were prepared from buffycoats of healthy donors by Histopaque-1077 (Sigma, 10771) densitygradient centrifugation at 250×g at room temperature for 30 min. Theintermediate layer containing PBMCs was collected and cells washed withPBS with 2 mM EDTA three times. Human monocytes were isolated from PBMCsby magnetic separation using human Classical Monocyte Isolation Kit(Miltenyi, 130-117-337) according to the manufacturer's instructions.2.5×10⁵ monocytes were cultured in 24-well plates in RPMI 1640 medium(Gibco, 11875093) containing 10% human AB serum, 10% FBS, L-glutamine,penicillin, and streptomycin at 37° C. with 5% CO2. Monocytes weretreated for 24 hr with 1 μg/mL Z10 or IgG control and supernatant washarvested for cytokine analysis. To differentiate M1 or M2 macrophages,monocytes were incubated with GM-CSF (100 ng/mL; Miltenyi, 130-093-864)or M-CSF (50 ng/mL; Miltenyi, 130-096-485) for 7 days, respectively, andthe effect of Z10 on differentiation was assessed by culturing in thepresence of 0.3 to 1 μg/mL Z10 or IgG throughout the whole time ofdifferentiation. To determine the effect on cytokine production of M2macrophages, the culture medium was replaced by serum-free X-Vivol5medium (Lonza, 04-418Q) with M-CSF (50 ng/mL) on Day 5 ofdifferentiation and treated with 1 μg/mL Z10 or IgG for 48 hrs, asindicated. To measure cytokines, cell culture supernatants werecollected and sent to Eve Technologies, Canada for HumanCytokine/Chemokine Array (HD42) analysis.

Analysis of Surface Molecules on Macrophages

Polarized macrophages were harvested with Cell Dissociation Buffer(ThermoFisher), washed twice, strained through a 70 m filter, andresuspended in FACS buffer (2% BSA, 10 mM EDTA, 25 mM HEPES, 0.1% sodiumazide in Ca/Mg-free PBS) containing 10% human AB serum. The cells werestained with fluorophore-conjugated antibodies against MERTK (BV421),CD86 (PE), and CD163 (APC) for 20 min on ice, and then with FixableViability Dye (eFluor 780) for 20 min on ice. Fluorescence signals wereacquired with a flow cytometer (BD LSR Fortessa), and analyzed withFlowJo and GraphPad Prism. Single color control samples were used tocreate a compensation matrix.

Proliferation Assay

SKMEL5 cells were seeded at 2,000 cells/well in a 96 well assay platewith Z10 or IgG control at concentrations of 0.1, 0.3, 0.9, 2.7, 8.1,and 24.3 μg/mL. After 72 hr of culture, cell proliferation wasdetermined by WST-8 Cell Proliferation Assay Kit (Cayman Chemical,10010199). The cells were incubated with WST-8 mixture for 1 hr and theoptical density was read at 450 nm using GloMax Multi Detection PlateReader (Promega). Background values from no cell control wells weresubtracted, and the means of triplicate wells were plotted usingGraphPad Prism.

Colony Formation Assay

SKMEL5 cells were plated at 500 cells/well in 6-well plates. The cellswere treated with 0.3 or 1 μg/mL of IgG control or Z10 in triplicates.After 7 days of culture, the media was exchanged with fresh mediacontaining the articles at the same concentration. The cells werestained on day 12. The wells were first washed once with PBS and thenstained with 2 mL of 6% glutaraldehyde (v/v), 0.5% crystal violet (w/v)in PBS for 30 minutes at room temperature. The plates were then rinsedtwice by immersing in a container filled with tap water. They then wereallowed to dry at room temperature. Once the plates were completely dry,the number of colonies of more than 50 cells were counted by microscopicobservation. The results were plotted as a bar graph using GraphPadPrism.

Western Blots

SKMEL5 cells were cultured at 3×10⁵ cells/well in 6-well platesovernight, and treated with Z10 at 0.01, 0.03, 0.1, 0.3, 1, 3, and 10μg/mL for 24 hr, or at 0.3 μg/mL for 10 min, 30 min, 2 hr, 4 hr, 8 hr,24 hr, 48 hr, and 72 hr. For GAS6 stimulation studies, SKMEL5 cells werecultured at 1×10⁵ cells/well in 24-well plate overnight, treated with0.3 μg/mL Z10 or IgG control for 2 hr, then stimulated with 200 nM GAS6(R&D Systems, 885-GSB-050) for 10 min.

Whole-cell lysates were prepared in RIPA buffer (Sigma, 20-188)containing protease inhibitor (Sigma, 4693159001) and phosphataseinhibitor (Sigma, P2850 and P5726). Protein concentrations were measuredusing BCA Protein Assay Kit (ThermoFisher, 23225). Equal protein amountswere loaded on 4-20% Tris-Glycine gel (Bio-Rad, 4561094), thentransferred to PVDF membranes (Bio-Rad, 1620177). After blocking with 5%BSA, membranes were probed with primary antibodies for 1 hr at roomtemperature. Bound antibodies were detected with HRP-conjugatedsecondary antibodies (Sigma, AP187P and AP181P) and visualized by ECLWestern Blotting Substrate (ThermoFisher, 32106), using ChemiDoc ImagingSystems (Bio-Rad).

Affinity Measurements by Competition ELISA of hMER, mMER, hAxl andhTyro3

Ninety-six well Nunc MaxiSorp flat-bottom plates (ThermoFisher,44-2404-21) were coated with 0.01 ug/mL hMER recombinant protein (RnDSystems, 891-MR-100) overnight at 4° C. Plates were washed three timeswith PBS 0.1% Tween and blocked with 3% non-fat dry skim milk in PBS for2 h.

During the saturation step, dilutions of the antigen hAxl (RnD Systems,154-AL-100), hTyro3 (RnD Systems, 859-DK-100) or mMER (RnD Systems,591-MR-100)] were prepared at 50 nM as the highest concentration, andserially diluted 1:1 to 0.098 nM in PBS. Each dilution of the antigenwas incubated with 150 uL of 6 nM of the MERTK (z10) antibody at roomtemperature for 1 h. Then, the saturated plate was washed three timeswith PBS 0.1% Tween and the antigen-antibody mixture was added to thewells in duplicate and incubated for 1 h at room temperature. The platewas then washed three times with PBS 0.1% Tween and incubated withanti-human Alkaline Phosphatase antibody (Sigma, SAB 3701248) at adilution of 1:3000 in PBS for 1 h at room temperature. After washingthree times with PBS 0.1% Tween, 100 uL of Alkaline Phosphatase Yellow(pNPP) liquid substrate system (Sigma, P7998) was added in each well andincubated for 70 min at room temperature. Plates were read at 405 nm andat 570 nm, to correct plate inconsistencies, using Glomax DiscoverSystem from Promega The absorbance was plotted as a function of antigenconcentration and the Kd was calculated using GraphPad Prism 7.0c.

6.2.2. Results

MERTK expressing melanoma cells (SKMEL5) were incubated with increasingconcentrations (0.002-13.5 μg/mL, (0.013-90 nM)) of APC-labeled z10antibody and then assessed by flow cytometry for APC MFI. As shown inFIG. 3A, the z10 antibody binds to human MERTK expressing melanoma cellswith high affinity (EC₅₀=6.7 nM). SPR measurements also demonstrate thatthe z10 antibody has high affinity for human MERTK (K_(D)=3 nM).

The z10 antibody specifically binds to human MERTK and does not bind tothe extracellular domain of human Axl, human Tyro3 or murine MERTK, asshown in FIGS. 4A-4D. An equilibrium between the z10 antibody (0.6 nM)and human MERTK, human Axl, human Tyro3 and murine MERTK extracellulardomains (50 nM-0.1 nM) was established before binding to solid-phaseantigen (human MERTK). Bound z10 antibody was labeled with alkalinephosphatase (AP) secondary antibody and developed for ELISA.

The z10 antibody was shown to induce human MERTK degradation of cancercells (see FIGS. 5A-5B). In particular, substantial degradation of humanMERTK was observed in SKMEL5 cells after 24 hours using concentrationsof 0, 0.03, 0.1, 0.3 and 1 μg/mL of the z10 antibody. Further,substantial degradation of human MERTK on SKMEL5 cells was observedafter 4 hours using 0.3 μg/mL of the z10 antibody.

In addition, the z10 antibody was demonstrated to induce degradation ofhuman MERTK of in vitro differentiated M2 macrophages (see FIGS. 6A-6B).Approximately 4 hours after in vitro differentiated M2 macrophages weretreated with 0.3 μg/mL of the z10 antibody, a decrease in more than 50%of the human MERTK was detected by Western Blot analysis (see FIGS.6A-6B). Even greater decreases in human MERTK on in vitro differentiatedM2 macrophages were detected after 8 hours of treatment with 0.3 μg/mLof the z10 antibody (see FIGS. 6A-6B).

The z10 antibody seems to degrade human MERTK on SKMEL5 human cancercells via internalization of the human MERTK and subsequent traffickingto the lysosome. As shown in FIGS. 7A-7B, after 24 h of incubation ofSKMEL5 cells with z10 antibody, the majority of the MERTK signal isfound in the lysosome and only minimal signal (<20%) is detected on thecell surface.

The z10 antibody prevents Gas6-induced AKT phosphorylation in humanMERTK-expressing SKMEL5 cells (see FIGS. 8A-8C). In particular, the AKTphosphorylation induced by Gas6 is blocked by treating cells with thez10 antibody. SKMEL5 cells were incubated with 0.3 μg/mL of the z10antibody or IgG control for 2 hours, followed by Gas6 treatment at 200nM for 10 minutes or buffer. As shown in FIG. 8A-8C, the incubation ofSKMEL5 cells with the z10 antibody prior to incubation with Gas6 reducesthe level of AKT phosphorylation induced by Gas6. FIG. 8B shows thathuman MERTK levels in the total lysate decreased after treatment withthe z10 antibody.

As shown in FIGS. 9A-9D, the z10 antibody inhibits colony formation ofcancer cells. 500 SKMEL5 cells were cultured on 6 well plates for 12days in the presence of control IgG or the z10 antibody (0.3 and 1μg/mL). Colonies of greater than 50 cells were counted. The number ofcolonies counted when the SKMEL5 cells were incubated with the z10antibody was significantly reduced relative to the colonies counted whenthe cells were incubated with control IgG.

The z10 antibody induced cytokine responses in M2 macrophages and CD14+monocytes (see FIG. 10). In particular, M2 macrophages were incubatedwith 1 μg/mL of the z10 antibody or control IgG for 48 hours and theexpression of certain cytokines was assessed. CD14+ monocytes wereincubated with 1 μg/mL of the z10 antibody or control IgG for 24 hoursand the expression of certain cytokines was assessed. Relative tocontrol IgG, the z10 antibody incudes increases in the expression ofcertain cytokines by M2 macrophages and CD14+ monocytes (see FIG. 10).M2 macrophages have been shown to suppress anti-tumor responses (see J.Immunol. Cancer 5(1):53 (2017) and human MERTK expression is mostlyrestricted to M2 macrophages (Crittenden et al. Oncotarget. 2016;7:78653-78666). Thus, the z10 antibody may have an anti-cancer effectindirectly in cancer cells and makes the z10 antibody useful fordelivery of toxic agents to cancer cells. In addition, human MERTK asbeen found to be expressed by various cancer cell lines (see entry forMERTK in The Human Protein Atlas, available from www.proteinatlas.org[accessed Feb. 26, 2019]). Thus, the z10 antibody, which has highaffinity and specificity for human MERTK induces cytokine responses byimmune cells and inhibits colony formation by cancer cells, hastherapeutic potential to treat a variety of cancers.

In addition, the EC₅₀s of antibodies z11 and z13 were determined using acell-based assay similar to that described in Section 6.2 above for z10.The z11 antibody has an EC₅₀ of 7.9 nM for human MERTK, while the z13antibody has an EC₅₀ of 7.6 nM for human MERTK.

6.3. Example 3: Effect of z10 on Cancer Cell Viability and Survival

6.3.1. Materials and Methods

Viability Assay

One thousand SKMEL5 cells and RPMI8226 cells were plated in 96-wellplate wells and cultured in 100 μL RPMI-1640 medium (ATCC) supplementedwith 10% FBS and Penicillin/Streptomycin containing either IgG control(BioXCell, BE0092) or z10 at 3 and 30 μg/mL. On day 4 (SKMEL5) or day 6(RPMI8226), 100 μL of CellTiter-Glo 2.0 Cell Viability Assay reagent(Promega, G9241) was added to each well, incubated for 10 mins and theluminescence signal was detected with a GloMax Discover MicroplateReader (Promega). The mean relative luminometer units (RLU) fromduplicate wells were plotted with GraphPad Prism.

Metastasis Assays

MDA-MB-231 LM2 TR triple negative breast cancer cells (received fromProf. Sohail Tavazoie, Rockefeller University, NYC) harboring aluciferase reporter gene (Minn et al., Nature. 2005 Jul. 28; 436(7050):518-524) were cultured in D10F culture medium (DMEM Life Technologies,11965-092), 10% FBS (Sigma, F4135-500 ml), 1% NEAA (Fisher, 11140-050),1% sodium pyruvate (Fisher, 11360-070), 1% Hepes (Fisher, 15630-080) and1% Pen/Strep (Fisher, 15140-122). On the day of injection, cells werewashed with DPBS (Fisher, 14190-144) and detached using 3 mL of 0.25%Trypsin-EDTA (Fisher, 25200-056) per 15 cm cell culture dish for 5 minin a 37° C. cell culture incubator. Detached cells were resuspended in 5mL of D10F, counted using a hemocytometer and centrifuged at 220×g for 5min at room temperature. The pellet was resuspended in DPBS at aconcentration of 500,000 cells/mL. 8-week-old NSG female mice (Jackson,005557) were exposed to a small animal heat lamp (MorganvilleScientific, HL0100) for 2-3 minutes. 100 μl of the cell suspension(50′000 cells) was injected into the dilated lateral tail vein of female8-week old NSG mice. IgG isotype control (BioXCell, BE0092) or z10 wasprepared in sterile 0.9% NaCl solution and administered i.p. every 3.5days, starting on the day of MDA-MB-231 LM2 cell injection. IgG isotypecontrol was dosed at 10 mg/kg and z10 was dosed at 1, 3, 10, and 30mg/kg. Lung colonization was monitored using IVIS imaging upon i.p.administration of 150 mg/kg D-luciferin (Thermofisher Scientific,Waltham, Mass.). The luminescent data for each time point was normalizedto day 0, or the day of tumor injection, and plotted using Prism(Graphpad Software, La Jolla, Calif.). Mann-Whitney U-test, one tailedwas used to assess significance of the metastasis as measured bybioluminescence imaging.

6.3.2. Results:

RPMI8226 multiple myeloma or SKMEL5 melanoma cells were cultured in thepresence of either IgG control or z10, respectively. On day 6 (RPMI8226)or day 4 (SKMel5), viability was assessed using CellTiter-Glo 2.0 CellViability Assay. FIGS. 11A and 11B show that z10 inhibits cell survivalin RPMi8226 and SKMEL5 cells, respectively.

50,000 MDA-MB-231-LM2 TNBC cells were injected into the tail vein of NSGmice. Treatment started on the day of tumor cell inoculation, twice/weeki.p. for the duration of the study. FIG. 12A shows tumor reduction inmice treated with z10. Data are quantified in FIG. 12B.

6.4. Example 4: Activity of Antibody-Drug Conjugates Comprising aHumanized Anti-MERTK Antibody

This example demonstrates that antibody-drug conjugates comprising ahumanized anti-MERTK antibody lead to MERTK internalization anddegradation, and inhibit the growth of cancer cells in vitro and invivo.

6.4.1. Materials and Methods

Synthesis of IgG1-Mc-Vc-PABC-MMAE (Also Termed Herein “IgG-MMAE”)

mc-vc-PABC-MMAE: 6317.7 μL of IgG₁ isotype control (11.08 mg/mL, WuXi,562) was incubated with 432.6 μL of Tris (2-carboxyethyl) phosphine(TCEP) (2.5 mM, Sigma, 1002068588) in 40 mM PB pH 7.0 solution (Sigma,101943380 and 101900512) at a final volume of 12.6 mL for 3 h at 37° C.with gentle rotation. Then, 830.5 μL Dimethylacetamide (DMA) (Sigma,ARK2190) and 569.5 μL of mc-vc-PABC-MMAE (10 mg/mL, Levena Biopharma,LN101-006) in DMA were added for a final DMA content of 10% v/v andmaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethylauristatin E (mc-vc-PABC-MMAE)/mAb ratio of 9. The reaction mixture wasincubated at 22° C. for 1 h with gentle rotation.

During the reaction, a 40 KD spin desalting column was equilibrated withPBS pH 7.2. This desalting column was used for the buffer exchange andpurification of the final product. The product was stored in 20 mMHistidine, pH 5.5. Protein concentration of this product was determinedby absorbance at 280 nm with EC280 as 1.61. Purity and aggregation levelof the product where determined by size-exclusion high performanceliquid chromatography (SEC-HPLC) with the conditions shown in Table 35below. Aggregation level of this product was 0.57% and monomeric purityof the product was 99.43%. Drug to antibody ratio (DAR) of the productwas measured with HIC-HPLC as 4.06.

Synthesis of z10-mc-vc-PABC-MMAE (also termed herein “z10-MMAE”)

9002.8 μL of z10 (7.22 mg/mL) was incubated with 410.535 μL TCEP (2.5mM) in 40 mM PB pH 7.0 solution at a final volume of 11.7 mL for 3 h at37° C. with gentle rotation. Then, 771.2 pL DMA and 528.76 μLmc-vc-PABC-MMAE (10 mg/mL) in DMA were added for a final DMA content of10% v/v and mc-vc-PABC-MMAE/mAb ratio of 9. The reaction mixture wasincubated at 4° C. for 22 h with gentle rotation. During the reaction, a40 KD spin desalting column was equilibrated with PBS pH 7.2. Thisdesalting column was used for the buffer exchange and purification. Theproduct was stored in 20 mM Histidine, pH 5.5. Protein concentration ofthis product was determined by absorbance at 280 nm with EC280 as 1.53.Purity and aggregation level of the product were determined bysize-exclusion high performance liquid chromatography (SEC-HPLC) withthe conditions shown in Table 35 below. Monomeric purity of the productwas 100%. DAR of the product was determined with HIC-HPLC as 3.88.

Synthesis of IgG1-CL2A-SN-38 (Also Termed Herein “IgG-SN-38”)

6272 μL of IgG1 isotype control (11.16 mg/mL, WuXi, 562) was incubatedwith 8 eq TCEP (769 μL, 5 mM) in PB pH 7.0 solution in a total volume of12.6 mL. The reaction was incubated at 37° C. for 3 h with gentlerotation. After that, the reaction mixture was cooled to 4° C. over anice bath followed by the addition of 327 μL of DMA and 1073 μL ofCL2A-SN-38 (10 mg/mL, Levena Biopharma, LN291-60) in DMA. The mixturewas kept at 4° C. for 1 h. During the reaction, a 40 KD spin desaltingcolumn was equilibrated with acetic acid pH 6.0. This desalting columnwas used for the buffer exchange and purification of the final product.The product was stored in 20 mM acetic acid pH 6.0. Proteinconcentration of this product was determined by the absorbance at 280 nmwith EC280 as 1.88 and reference wavelength at 700 nm. Aggregation andpurity of the product was determined with SEC-HPLC with the conditionsshown in Table 36 below. Aggregation level of this product was 1.14% andpurity 99.14%. DAR of the product was determined with LC-MS as 7.78.

Synthesis of z10-CL2A-SN-38 (Also Termed Herein “z10-SN-38”)

9002 μL of z10 (7.22 mg/mL) was incubated with 8 eq TCEP (714 μL, 5 mM)in PB pH 7.0 solution in a total volume of 11.7 mL. The reaction mixturewas incubated at 37° C. for 3 h with gentle rotation. After that, thereaction mixture was cooled to 4° C. over an ice bath followed by theaddition of 90 μL DMA and 1210 μL of CL2A-SN-38 (10 mg/ml, LevenaBiopharma, LN291-60) in DMA. The mixture was kept at 4° C. for 1 h.During the reaction, a 40 KD spin desalting column was equilibrated withacetic acid pH 6.0. This desalting column was used for the bufferexchange and purification of the final product. The product was storedin 20 mM acetic acid pH 6.0. Protein concentration of this product wasdetermined by the absorbance at 280 nm with EC280 as 1.82. Monomericpurity of the product was determined with SEC-HPLC with the conditionsshown in Table 36 below. Thus aggregation level of this product is 1.14%and the monomeric purity of the product is 98.86%. DAR of the productwas determined with LC-MS as 7.49.

Determination of Purity and Aggregation Levels

Tables 35 and 36 present the SEC-HPLC parameters by which purificationand aggregation of the antibody-drug conjugates were determined.

TABLE 35 SEC-HPLC for purity and aggregation level of Ab-mc-vc-PABC-MMAEproduct HPLC parameters Equipment Agilent 1260 series HPLC Column TSKgelG3000SWXL 7.8 mm*300 mm, 5 μm particle size Column Temp. 25° C. Mobilephase A: 200 mM KPO₄, 250 mM KCl, 15% IPA, PH 7.0 Flow rate 0.75 mL/minSampler Temp. 4° C. Injection volume 11 μL Detection 280 nm wavelengthTime (min) A % B % Gradient 0 100 25 18 100 40

TABLE 36 SEC-HPLC for purity and aggregation level of Ab-CL2A-SN-38 HPLCparameters Equipment Agilent 1260 series HPLC Column TSKgel G3000SWXL7.8 mm*300 mm, 5 μm particle size Column Temp. 25° C. Mobile phase A:200 mM KPO₄, 250 mM KCl, 15% IPA, PH 7.0 Flow rate 0.75 mL/min SamplerTemp. 4° C. Injection volume 11 μL Detection 280 nm wavelength Time(min) A % B % Gradient 0 100 25 18 100 40

Internalization Assay

IgG control (R&D 1-001-A), z10, z10-MMAE, and z10-SN-38 were labeledwith pHrodo iFL Red, a pH-sensitive dye, using pHrodo iFL Red MicroscaleProtein Labeling Kit (ThermoFisher, P36014) according to themanufacturer's instructions. Internalization of the antibody wasdetermined by flow cytometry detecting pHrodo fluorescence, which isminimal at neutral pH and maximal in acidic environments, such as thelysosome.

A hundred thousand SKMEL5 cells per well were plated on 24-well platesin 1 mL RPMI-1640 medium (ATCC) supplemented with 10% FBS andPenicillin/Streptomycin and incubated with 10 nM pHrodo-labeled IgG,z10, z10-MMAE, or z10-SN-38, respectively, for 3 or 6 hrs. The cellswere trypsinized, strained through a 70 μm filter, and resuspended inFACS buffer (2% BSA, 10 mM EDTA, 25 mM HEPES, 0.1% sodium azide inCa/Mg-free PBS) containing Human TruStain FcX blocking reagent(Biolegend, 422301). Cells were sequentially stained withBV421-conjugated antibodies against MERTK to detect surface MERTK levels(Biolegend, 367603) for 20 min on ice, and then stained with FixableViability Dye (eFluor 780) (Thermofisher, 65-0865-14) for 20 min on ice.

The Mean Fluorescence Intensities (MFI) of pHrodo and BV-421 wereanalyzed with LSRFortessa flow cytometer (BD Biosciences) andcompensated data were plotted with GraphPad Prism. The MFI of pHrodo wasnormalized with Degree of Labeling (DOL) of pHrodo-labeled IgG, z10,z10-MMAE and z10-SN-38. DOL was calculated from A280 and A560 absorbanceof the protein after labeling, according to the formula provided by themanufacturer.

Cancer Cell Line Viability Assay

Two hundred SKMEL5 cells or one thousand RPMI8226 cells per well wereplated in 96-well plate wells and cultured in 100 μL RPMI-1640 medium(ATCC) supplemented with 10% FBS and Penicillin/Streptomycin containingeither IgG control (BioXCell, BE0092), IgG1-MMAE, z10-MMAE, IgG1-SN-38or z10-SN-38, respectively, at 0.015, 0.046, 0.137, 0.412, 1.24, 3.70,11.1, 33.3, and 100 nM. On day 7, 100 μL of CellTiter-Glo 2.0 CellViability Assay reagent (Promega, G9241) was added to each well,incubated for 10 mins and the luminescence signal was detected with aGloMax Discover Microplate Reader (Promega). The mean relativeluminometer units (RLU) from duplicate wells were plotted with GraphPadPrism.

Macrophage Viability Assay

Peripheral blood mononuclear cells (PBMCs) were prepared from buftycoats of healthy donors by Histopaque-1077 (Sigma, 10771) densitygradient centrifugation at 250×g at room temperature for 30 min. Theintermediate layer containing PBMCs was collected and cells were washedwith PBS/2 mM EDTA three times. Human monocytes were isolated from PBMCsby magnetic separation using human Classical Monocyte Isolation Kit(Miltenyi, 130-117-337) according to the manufacturer's instructions.Eighty thousand monocytes per well were cultured on 96-well plates in150 μL PBMC medium (RPMI 1640 medium (Gibco, 11875093) containing 10%/3human AB serum, 10% FBS, L-glutamine, penicillin, and streptomycin) at37° C. with 5% CO₂. Monocytes were treated with GM-CSF (20 ng/mL;Biolegend, 766102) or M-CSF (50 ng/mL; Biolegend, 574804) for 8 days todifferentiate to M1 or M2 macrophages, respectively. Differentiatedmacrophages were then treated with IgG control (2 or 20 nM; BioXCell,BE0092), z10 (2 or 20 nM), IgG1-MMAF (1 or 10 nM), z10-MMAE (1 or 10nM), IgG1-SN-38 (0.03 or 0.3 nM), z10-SN-38 (0.03 or 0.3 nM) or 1 μM ofthe small molecule MERTK inhibitor UNC-1062 (Aobious, AOB4488), in 100μL GM-CSF or M-CSF containing differentiation medium. On day 4, 100 μLof CellTiter-Glo 2.0 Cell Viability Assay reagent (Promega, G9241) wasadded to each well, incubated for 10 mins and the luminescence signalwas detected with a GloMax Discover Microplate Reader (Promega). Themean relative luminometer units (RLU) from duplicate wells were plottedwith GraphPad Prism.

Western Blot

SKMEL5 cells and RPMI8226 cells were cultured and maintained inRPMI-1640 medium (ATCC) supplemented with 10% FBS andPenicillin/Streptomycin. Whole-cell lysates were prepared in RIPA buffer(Sigma, 20-188) containing protease inhibitor (Sigma, 4693159001) andphosphatase inhibitor (Sigma, P2850 and P5726). Protein concentrationswere measured using BCA Protein Assay Kit (ThermoFisher, 23225). Equalprotein amounts (30 μg) were loaded on 4-20% Tris-Glycine gel (Bio-Rad,4561094), then transferred to PVDF membranes (Bio-Rad, 1620177). Afterblocking with 5% BSA, membranes were probed with primary antibodies for1 hr at room temperature in PBST (0.1% Tween-20 in PBS) with 1% BSA.After 3 wash steps in PBST with 1% BSA, HRP-conjugated secondaryantibodies were added in PBST with 1% BSA and incubated for 1 h (Sigma,AP187P and AP181P). The blot was washed with PBST and developed usingECL Western Blotting Substrate (ThermoFisher, 32106), using ChemiDocImaging Systems (Bio-Rad). The signal was normalized to tubulin, used asinternal control. The Following primary antibodies were used in Westernblot assays; MERTK (abcam, ab52968), phospho MERTK (abcam, ab14921), AXL(CST, 8661), Tyro3 (CST, 5585), AKT (abcam, ab32505), phospho AKT(abcam, ab81283), Tubulin (Sigma, T5168).

Metastasis Assays

MDA-MB-231 LM2 TR triple negative breast cancer cells (received fromProf. Sohail Tavazoie, Rockefeller University, NYC) harboring aluciferase reporter gene (Minn et al., Nature. 2005 Jul. 28; 436(7050):518-524) were cultured in D10F culture medium (DMEM Life Technologies,11965-092, 10% FBS Sigma, F4135-500 ml, 1% NEAA Fisher, 11140-050, 1%sodium pyruvate Fisher, 11360-070, 1% Hepes Fisher, 15630-080 and 1%Pen/Strep Fisher, 15140-122. On the day of injection, cells were washedwith DPBS (Fisher, 14190-144) and detached using 3 mL of 0.25%Trypsin-EDTA (Fisher, 25200-056) per 15 cm cell culture dish for 5 minin a 37° C. cell culture incubator. Detached cells were resuspended in 5mL of D1F, counted using a hemocytometer and centrifuged at 220×g for 5min at RT. The pellet was resuspended in DPBS at a concentration of500,000 cells/mL. 8-week-old NSG female mice (Jackson, 005557) wereexposed to a small animal heat lamp (Morganville Scientific, HL0100) for2-3 minutes. 100 μL of the cell suspension (50′000 cells) was injectedinto the dilated lateral tail vein of female 8-week old NSG mice.IgG-MMAE isotype control and z10-MMAE were prepared in sterile PBS andadministered i.p. on Day 0, 3, 10, 18, 33, and 41 after MDA-MB-231 LM2cell injection. IgG-MMAE and z10-MMAE were initially dosed at 3 mg/kg,and reduced to 2 mg/kg on Day 18, and to 1 mg/kg on Day 33.

Lung colonization was monitored using IVIS imaging upon i.p.administration of 150 mg/kg D-luciferin (Thermofisher Scientific,Waltham, Mass.). The luminescent data for each time point was normalizedto day 0, or the day of tumor injection, and plotted using Prism(Graphpad Software, La Jolla, Calif.). Mann-Whitney U-test, one tailedwas used to assess significance of the metastasis as measured bybioluminescence imaging.

Affinity measurements by competitive ELISA

Ninety-six well Nunc MaxiSorp flat-bottom plates (ThermoFisher,44-2404-21) were coated with 0.01 μg/mL hMER recombinant protein (R&DSystems, 891-MR-100) overnight at 4° C. Plates were washed three timeswith PBS 0.1^(Q)% Tween and blocked with 3% non-fat dry skim milk in PBSfor 2 h. During the pre-incubation of the plate, hMER recombinantprotein in PBS at various concentrations (50, 25, 12.5, 6.25, 3.13,1.56, 0.78, 0.39, 0.20, 0.10, 0.05 and 0 nM) were equilibrated with 0.3nM z10, z10-MMAE, or z10-SN-38, respectively at room temperature for 1h. Then, the hM ER-coated plate was washed three times with PBST (0.1%Tween-20 in PBS) and the antigen-antibody mixture was added to the wellsin duplicates and incubated for 1 h at room temperature. The plate waswashed three times with PBST and incubated with AlkalinePhosphatase-conjugated anti-human IgG antibody (Sigma, SAB3701248) at adilution of 1:3000 in PBS for 1 h at room temperature. After washingthree times with PBST, 100 μL of Alkaline Phosphatase Yellow (pNPP)liquid substrate (Sigma, P7998) was added to each well and incubated for70 min at room temperature. Plates were measured for absorbance at 405nm and 560 nm using GloMax® Discover Microplate Reader (Promega). The405 nm absorbance was normalized with 560 nm absorbance. The mean valuesfrom duplicate wells were plotted as a function of antigen concentrationand the Kd was determined by nonlinear regression analysis with GraphPadPrism 7.0c.

6.4.2. Results

z10-mc-vc-PABC-MMAE (z10-MMAE) and IgG1-mc-vc-PABC-MMAE (IgG-MMAE) weresynthesized, with their structures shown in FIG. 13A where the Ab(antibody portion) was z10 and control IgG, respectively. z10-CL2A-SN-38(z10-SN-38) and IgG₁-CL2A-SN-38 (IgG-SN-38) were synthesized, with theirstructures shown in FIG. 13B where the Ab (antibody portion) was z10 andcontrol IgG, respectively.

Purity of the antibody-drug conjugates was assessed by SEC-HPLC. FIGS.14A and 14B show SEC-HPLC results for z10-MMAE and z10-SN-38,respectively. For both conjugates, no or minimal aggregation wasmeasured, indicating high purity of the conjugates.

To assess the binding affinity of the antibody-drug conjugates and z10,hMER recombinant protein (50 nM-0.05 nM) was equilibrated with 0.3 nMz10, z10-MMAE, or z10-SN-38, respectively before addition to ELISAplates pre-coated with hMER recombinant protein. Antibody binding wasdetected using AP-conjugated anti-human IgG secondary antibody anddeveloped using a GloMax microplate reader. FIG. 15 shows that z10,z10-MMAE and z10-SN-38 bind to human MER with Kd values of 0.17 nM,0.081 nM and 0.021 nM, respectively.

SKMel5 cells were incubated with 6.7 nM of pHrodo-labeled z10, z10-MMAE,z10-SN-38, or IgG control. Surface MERTK was stained with aBV421-conjugated MERTK antibody. Binding was measured by flow cytometry.The data shown in FIG. 16A demonstrate that z10 as well as the z10-MMAEand z10-SN-38 conjugates induce MERTK internalization. The data shown inFIG. 16B demonstrate that z10 as well as the z10-MMAE and z10-SN-38conjugates induce MERTK degradation.

SKMel5 or RPMI8226 cells were incubated with IgG control, IgG-MMAE,z10-MMAE, IgG-SN-38 or z10-SN-38 for 7 days. Cell viability was measuredusing CellTiterGlo. The data shown in FIGS. 17A and 17B demonstrate thatz10-MMAE decreased viability of SKMel5 melanoma and RPMI8226 multiplemyeloma cells, respectively, compared to the viability of these cellsfollowing incubation with IgG-MMAE or IgG control. The data shown inFIGS. 17C and 17D demonstrate that z10-SN-38 decreased viability ofSKMel5 melanoma and RPMI8226 multiple myeloma cells, respectively,compared to the viability of those cells incubated with IgG-SN-38 or IgGcontrol. FIG. 17E shows the expression of MERTK, phospho MERTK, AXL,Tyro3, AKT, phospho AKT, and tubulin in whole cell lysates prepared fromSKMel5 and RPMI8226 cells.

50,000 MDA-MB-231-LM2 TNBC cells were injected into the tail vein of NSGmice. Treatment started on the day of tumor cell inoculation and lungmetastatic colonization was monitored by bioluminescence imaging. FIG.18A shows that z10-MMAE inhibits lung colonization of MDA-MB-231triple-negative breast cancer cells compared to IgG-MMAE in vivo. Dataare quantified in FIG. 18B.

M2 macrophages were differentiated by culturing 8×10⁴ monocytes in PBMCmedium with M-CSF (50 ng/mL) for 8 days. M1 macrophages weredifferentiated by culturing 8×10⁴ monocytes in PBMC medium with GM-CSF(20 ng/mL) for 5 days, then cultured with GM-CSF (20 ng/mL), IFNg (20ng/mL), IL-6 (20 ng/mL), and LPS (10 pg/mL) for additional 3 days. M2and M1 macrophages were treated with indicated antibodies or ADCs for 4days in differentiating medium and viability was assessed usingCellTiter-Glo 2.0 Cell Viability Assay. The data shown in FIG. 19Ademonstrate that neither z10 nor z10-MMAE nor z10-SN-38 nor the MERTKinhibitor UNC 1062 affect the viability of MERTK-expressing M1macrophages. The data shown in FIG. 19B demonstrate that neither z10 norz10-MMAE nor z10-SN-38 nor the MERTK inhibitor UNC 1062 affect theviability of MERTK-expressing M2 macrophages.

The foregoing is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the antibodies andmethods provided herein and their equivalents, in addition to thosedescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying figures. Such modificationsare intended to fall within the scope of the appended claims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

1. An antibody or an antigen-binding fragment thereof that specificallybinds to human Mer Tyrosine Kinase (MERTK), wherein the antibody orantigen-binding fragment comprises a heavy chain variable region (VH)and a light chain variable region (VL), wherein (i) the VH comprises theamino acid sequence of sequence of SEQ ID NO: 105 and the VL comprisesthe amino acid sequence of SEQ ID NO: 106; (ii) the VH comprises theamino acid sequence of sequence of SEQ ID NO: 107 or 108 and the VLcomprises the amino acid sequence of SEQ ID NO: 106; (iii) the VHcomprises the amino acid sequence of sequence of SEQ ID NO: 108 or 109and the VL comprises the amino acid sequence of SEQ ID NO: 106; or (iv)the VH comprises the amino acid sequence of sequence of SEQ ID NO: 110and the VL comprises the amino acid sequence of SEQ ID NO:
 120. 2.-4.(canceled)
 5. The antibody or antigen-binding fragment thereof of claim1, wherein said antibody is a monoclonal antibody.
 6. The antibody orantigen-binding fragment thereof of claim 1, wherein the antibody is animmunoglobulin comprising two identical heavy chains and two identicallight chains.
 7. The antibody or antigen-binding fragment thereof ofclaim 1, wherein the antibody comprises human-derived heavy and lightchain constant regions.
 8. The antibody or antigen-binding fragmentthereof of claim 1, wherein the antibody is an IgG.
 9. The antibody orantigen-binding fragment thereof of claim 1, wherein the antibodycomprises a portion of human-derived heavy and light chain constantregions.
 10. The antibody or antigen-binding fragment thereof of claim1, wherein the antigen-binding fragment is an Fab or F(ab′)2 fragment.11. A bispecific antibody comprising two different antigen-bindingregions, wherein one antigen-binding region comprises the antibody orantigen-binding fragment of claim 1, and wherein the otherantigen-binding region binds to an antigen of interest.
 12. Thebispecific antibody of claim 11, wherein the antigen of interest is animmune cell receptor or tumor-associated antigen.
 13. The bispecificantibody of claim 11, wherein the antigen of interest is CD3, PD-L1,LRP1, LPR8, TGF-β, ICOS, CD40, NKGD2, or TIGIT.
 14. An antibody-drugconjugate comprising: (a) an antibody moiety that is the antibody or anantigen-binding fragment thereof of claim 1; (b) one or more drugmoieties, each drug moiety being a cytotoxic agent, and (c) optionally alinker, wherein the cytotoxic agent is conjugated directly to theantibody moiety or is conjugated to the antibody moiety via the linker.15. The antibody-drug conjugate of claim 14, which has a molar ratio ofthe antibody moiety to the drug moiety that is between 1:3 and 1:12. 16.The antibody-drug conjugate of claim 14, which has a molar ratio of theantibody moiety to the drug moiety of 1:9.
 17. (canceled)
 18. (canceled)19. The antibody-drug conjugate of claim 14, wherein the cytotoxic agentis an auristatin, a maytansinoid, a pyrrolobenzodiazepine, anindolinobenzodiazepine, a calicheamicin, a camptothecin analogue, aduocarmycin, a tubulin inhibitor, a tubulysin or tubulysin analogue,amberstatin269, doxorubicin, SN-38, an antibiotic, an anthracycline, amicrotubule inhibitor, a spliceostatin, or a thailanstain.
 20. Theantibody-drug conjugate of claim 14, wherein the cytotoxic agent ismonomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). 21.The antibody-drug conjugate of claim 14, wherein the cytotoxic agent isDM1 or DM4.
 22. The antibody-drug conjugate of claim 14, wherein thecytotoxic agent is monomethyl auristatin E.
 23. The antibody-drugconjugate of claim 14, wherein the cytotoxic agent is SN-38.
 24. Theantibody-drug conjugate of claim 14, wherein the antibody-drug conjugatecomprises the linker, and the linker is a cleavable linker.
 25. Theantibody-drug conjugate of claim 14, wherein the antibody-drug conjugatecomprises the linker, and the linker is a non-cleavable linker.
 26. Theantibody-drug conjugate of claim 14, wherein the antibody-drug conjugatecomprises the linker, and the linker ismaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl.
 27. Theantibody-drug conjugate of claim 14, wherein the antibody-drug conjugatecomprises the linker, and the linker is CL2.
 28. The antibody-drugconjugate of claim 14, wherein the antibody-drug conjugate comprises thelinker, and the linker is CL2A.
 29. An isolated nucleic acid sequencecomprising a polynucleotide encoding the antibody or antigen-bindingfragment thereof of claim
 1. 30. An isolated nucleic acid sequencecomprising a polynucleotide encoding the VH of claim
 1. 31. An isolatednucleic acid sequence comprising a polynucleotide encoding the VL ofclaim
 1. 32. An ex vivo cell containing one or more polynucleotidesencoding the antibody or antigen-binding fragment of claim
 1. 33. Amethod of producing an antibody or antigen-binding fragment comprisingculturing the cell of claim 32 under conditions such that said one ormore polynucleotides are expressed by the cell to produce the antibodyor antigen-binding fragment encoded by the polynucleotides.
 34. A methodof producing the antibody-drug conjugate of claim 14, wherein saidlinker is not present, said method comprising: (a) conjugating thecytotoxic agent directly to the antibody moiety to produce theantibody-drug conjugate; and (b) purifying the antibody-drug conjugate.35. A method of producing the antibody-drug conjugate of claim 14,wherein said antibody-drug conjugate comprises said linker, said methodcomprising the following steps in the order stated: (a) conjugating thelinker directly to the antibody moiety to produce a linker-antibodymoiety; (b) conjugating the linker of the linker-antibody moietydirectly to the cytotoxic agent to produce the antibody-drug conjugate;and (c) purifying the antibody-drug conjugate.
 36. A method of producingthe antibody-drug conjugate of claim 14, wherein said antibody-drugconjugate comprises said linker, said method comprising the followingsteps in the order stated: (a) conjugating the linker directly to thecytotoxic agent to produce a linker-cytotoxic agent moiety; (b)conjugating the linker of the linker-cytotoxic agent moiety directly tothe antibody moiety to produce the antibody-drug conjugate; and (c)purifying the antibody-drug conjugate.
 37. A pharmaceutical compositioncomprising a therapeutically effective amount of the antibody orantigen-binding fragment of claim 1, and a pharmaceutically acceptablecarrier.
 38. A method of treating cancer in a subject in need thereof,comprising administering to said subject the pharmaceutical compositionof claim
 37. 39.-60. (canceled)
 61. An antibody-drug conjugatecomprising an antibody moiety comprising a heavy chain variable region(VH) and a light chain variable region (VL), wherein the VH comprisesthe amino acid sequence of sequence of SEQ ID NO: 105 and the VLcomprises the amino acid sequence of SEQ ID NO: 106, wherein theantibody moiety is conjugated to MMAE via an mc-vc-PABC linker.
 62. Anantibody-drug conjugate comprising an antibody moiety comprising a heavychain variable region (VH) and a light chain variable region (VL),wherein the VH comprises the amino acid sequence of sequence of SEQ IDNO: 105 and the VL comprises the amino acid sequence of SEQ ID NO: 106,wherein the antibody moiety is conjugated to SN-38 via the CL2A linker.63. (canceled)
 64. (canceled)
 65. A pharmaceutical compositioncomprising a therapeutically effective amount of the antibody-drugconjugate of claim 14, and a pharmaceutically acceptable carrier.
 66. Amethod of treating cancer in a subject in need thereof, comprisingadministering to said subject the pharmaceutical composition of claim65.